University of Groningen
Survival and cellular heterogeneity of epithelium in cultured mouse and rat precision-cut
intestinal slices
Biel, Carin; Bigaeva, Emilia; Hesse, Melanie; Bomers, Jordy J.M.; van Summeren, Kitty;
Teunis, Marc A.T.; Vaessen, Stefan; Ten Klooster, Jean Paul; Olinga, Peter
Published in:
Toxicology in Vitro
DOI:
10.1016/j.tiv.2020.104974
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Biel, C., Bigaeva, E., Hesse, M., Bomers, J. J. M., van Summeren, K., Teunis, M. A. T., Vaessen, S., Ten
Klooster, J. P., & Olinga, P. (2020). Survival and cellular heterogeneity of epithelium in cultured mouse and
rat precision-cut intestinal slices. Toxicology in Vitro, 69, [104974]. https://doi.org/10.1016/j.tiv.2020.104974
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Toxicology in Vitro
journal homepage:www.elsevier.com/locate/toxinvit
Survival and cellular heterogeneity of epithelium in cultured mouse and rat
precision-cut intestinal slices
Carin Biel
a,1, Emilia Bigaeva
a,b,1, Melanie Hesse
a, Jordy J.M. Bomers
a,c, Kitty van Summeren
d,
Marc A.T. Teunis
d, Stefan Vaessen
d, Jean Paul Ten Klooster
d, Peter Olinga
a,⁎aDepartment of Pharmaceutical Technology and Biopharmacy, University of Groningen, the Netherlands
bDepartment of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands cPROdermpath, Labor für Dermatohistopathology, Vreden, Germany
dResearch Centre for Healthy and Sustainable Living, Innovative Testing in Life Sciences and Chemistry, University of Applied Sciences, Utrecht, the Netherlands
A R T I C L E I N F O Keywords:
Precision-cut intestinal slices Small intestine
Ex vivo
Intestinal epithelial cells
A B S T R A C T
Precision-cut intestinal slices (PCIS) are used to study intestinal (patho)physiology, drug efficacy, toxicity, transport and metabolism ex vivo. One of the factors that limit the use of PCIS is a relatively short life-span. Moreover, culture-induced changes in cellular composition of PCIS remain largely uncharacterized. In this study, we demonstrated the epithelial cell heterogeneity in mouse and rat PCIS and its alterations during culture. In addition, we evaluated whether the presence of niche growth factors impacts the survival of PCIS epithelial cells. We showed that freshly prepared PCIS retained the main epithelial cell types, namely absorptive enterocytes, goblet cells, enteroendocrine cells, stem cells, transit-amplifying cells and Paneth cells. Once placed in culture, PCIS displayed progressive epithelial damage, and loss of these epithelial cell types. Cells comprising the in-testinal stem cell niche were especially sensitive to the damage, and the addition of niche growth factors ben-eficially affected the survival of stem cells and transit-amplifying cells in PCIS during culture. In conclusion, this study provides new insights into the dynamic changes in cellular composition of epithelium in cultured PCIS, paving the way to future toxicological and pharmacological studies in an informed and reliable ex vivo setting.
1. Introduction
Drug-induced gastro-intestinal toxicity is one of the major adverse effects of orally given medication. There is no drug classification based on the intestinal toxicity, since the mechanism of toxicity is often complicated and there is hardly a relation between the morphological appearance of the intestinal tissue and clinical symptoms (Scarpignato and Bjarnason, 2019). In vitro cell cultures as well as in vivo animal models have been developed to predict intestinal toxicity, metabolism and transport of xenobiotics. However, these models either lack the complexity of the intestinal tissue or appear impractical and have a low screening capacity (Li et al., 2016;Niu et al., 2013).
Precision-cut intestinal slices (PCIS) is an ex vivo model that is used to study intestinal disease, physiology, drug efficacy, toxicity, transport and metabolism (de Graaf et al., 2010;Li et al., 2016). Since PCIS are prepared from fresh tissue, the multicellular 3D tissue structure and polarized epithelium remain intact during the first hours of culture (van de Kerkhof et al., 2005). While PCIS can be obtained from different
species, including human, there are a number of reasons to use animal PCIS such as limited availability of human tissue, better reproducibility, established disease models and possibility for genetic alterations, among others. Mice and rats are the most used laboratory animals and therefore frequently used to generate PCIS (Iswandana et al., 2016a).
PCIS from mouse and rat intestinal tissue consist of all layers of the intestinal wall, including (sub)mucosa, muscularis externa and serosa (de Graaf et al., 2010). In vivo, the epithelial layer of the intestine is continuously renewed. At the villus tip, cells undergo apoptosis, while at the bottom of crypts, stem cells proliferate and their daughter cells differentiate into specialized cell types of the epithelial layer (Scoville et al., 2008). We previously showed that culturing of PCIS is associated with a decrease in viability and deterioration of structural integrity of intestinal tissue, which include damaged epithelial monolayer, flat-tening of the villi and loss of crypts (Bigaeva et al., 2019a). However, the epithelial cell heterogeneity and the survival of specialized cells in epithelium of PCIS during culture have never been described.
The aim of this study was to characterize the changes in integrity
https://doi.org/10.1016/j.tiv.2020.104974
Received 25 June 2020; Received in revised form 5 August 2020; Accepted 19 August 2020 ⁎Corresponding author.
E-mail address:p.olinga@rug.nl(P. Olinga). 1These authors contributed equally to this work.
Available online 21 August 2020
0887-2333/ © 2020 Elsevier Ltd. All rights reserved.
and cellular heterogeneity of epithelium in PCIS during culture. To this end, we determined the presence of main types of epithelial cells in mouse and rat PCIS before and during culture and investigated whether culturing affected biological processes, such as apoptosis, proliferation, inflammation and wound healing response. Furthermore, following our previous findings that the addition of growth factors of the intestinal stem cell niche may likely extend the life-span of mouse and rat PCIS (Bigaeva et al., 2019a), we investigated whether these growth factors improve the survival of epithelial cell subtypes during culture. This study provides new knowledge on the dynamic changes in cellular composition of epithelium ex vivo, and lays the foundation for future refined toxicological and pharmacological studies in a relevant ex vivo setting.
2. Materials & methods 2.1. Animals
Male, 8–10 weeks old, C57BL/6 J mice and male, 12–14 weeks old, Wistar rats, (Centrale Dienst Proefdieren, University Medical Center Groningen, Groningen, The Netherlands) were housed in a tempera-ture- and humidity-controlled room with a 12 h light/dark cycle, with water and food ad libidum. The experiments were approved by the Animal Ethical Committee of the University of Groningen (CCD number AVD105002017884) and were performed in accordance with the EU directive 2010/63/EU for animal experiments.
2.2. Preparation and incubation of precision-cut intestinal slices (PCIS) Mouse and rat intestines were harvested during a terminal proce-dure under isoflurane/O2 anesthesia, and immediately stored in ice-cold oxygenated Krebs-Henseleit buffer (KHB), containing 25 mMD
-glucose (Merck, Darmstadt, Germany), 25 mM NaHCO3 (Merck,
Darmstadt, Germany) and 10 mM HEPES (MP Biomedicals, Aurora, OH), pH 7.4. PCIS were prepared as previously described (de Graaf et al., 2010). Briefly, the jejunum was flushed with KHB and cut into segments. These segments were filled with 3% (w/v) agarose (Sigma-Aldrich, Saint Louis, USA) in 0.9% (w/v) NaCl solution at 37 °C, and then embedded in an agarose core-embedding unit. PCIS were prepared using the Krumdieck tissue slicer (Alabama Research and Development, Munford, USA) and collected in ice-cold KHB.
Mouse and rat PCIS (mPCIS and rPCIS, respectively) were cultured for different time periods in standard culture medium, i.e. Williams' Medium E (WME, Gibco™, Life Technologies, Bleiswijk, The Netherlands) supplemented with 25 mMD-glucose (Merck, Darmstadt,
Germany), 50 μg/mL gentamycin (Gibco™, Life Technologies, Bleiswijk, The Netherlands) and 2.5 μg/mL amphotericin-B (Gibco™, Life Technologies, Bleiswijk, The Netherlands). Slices were incubated in-dividually in 24- wells plates (mPCIS) or 12-wells plates (rPCIS), while shaking (90 rpm) at 37 °C in 80% O2/ 5% CO2. The culture medium was refreshed every 24 h. MPCIS were harvested directly after slicing and after 24, 48, 72, 96, and 120 h of incubation. RPCIS were harvested directly after slicing and after 3, 6, 24 and 48 h of incubation.
Alternatively, PCIS were incubated for 24 (rPCIS) or 96 (mPCIS) hours in media of different compositions (Table 1). Culture media were purchased at Gibco™ Life Technologies (Bleiswijk, The Netherlands) and supplemented with 10% (v/v) FBS (Sigma-Aldrich, Saint Louis, USA), 10% (v/v) Noggin, 10% (v/v) R-spondin-1 conditioned medium, 10% (v/v) Wnt3a conditioned medium, 50 ng/mL epithelial growth factor (EGF) (Gibco™, Life Technologies, Bleiswijk, The Netherlands), 1× N2/B27 (Life Technologies) and 10 mM HEPES (MP Biomedicals, Aurora, OH). Conditioned media (Noggin, R-spondin-1 and Wnt3a) were prepared as previously described (Bigaeva et al., 2019a). MPCIS were harvested after 96 h of incubation, while rPCIS were harvested after 24 h of incubation.
2.3. Determination of ATP and protein content
The viability of PCIS was determined by measuring the adenosine tri-phosphate (ATP)/per protein (de Graaf et al., 2010). PCIS were collected (n = 3 slices per condition) in 0.5 mL (mPCIS) or 1 mL (rPCIS) sonication solution (2 mM in EDTA 70% ethanol), snap-frozen and stored at −80 °C until measurement. The ATP and protein content was determined as previously described (Bigaeva et al., 2019a), using the ATP bioluminescence kit (Roche diagnostics, Manheim, Germany, cat. no. 11699695001) and the Lowry assay (Bio-Rad DC Protein Assay, Veenendaal, The Netherlands, cat. no. 5000122), according to manu-facturers' protocols.
2.4. Histomorphology and immunohistochemistry
After incubation, PCIS (n = 3 slices/condition/animal) were fixed for 24 h in 4% buffered formalin and dehydrated for at least 24 h in 70% ethanol at 4 °C. After processing, PCIS were embedded in paraffin and cut into 3–4 μm thick sections.
Overall morphological appearance of PCIS was assessed by hema-toxylin and eosin (H&E) staining. Obtained H&E stained tissue sections were then subjected to a blind histological scoring, which evaluated epithelial injury (shape and necrosis), villi preservation, crypt pre-servation and stromal injury (necrosis), according to an adapted his-tological scoring system. A higher score indicates a poorer morphology (Supplementary Table 1;Bigaeva et al., 2019a). The scoring was per-formed independently by four researchers (C.B., E.B., J.J.M.B. and M.H.). Periodic acid-Schiff (PAS) staining was performed in accordance with the standard histological protocol to determine the presence of mucin-producing goblet cells. To assess the amount of cell death, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit (Abcam, Cambridge, UK, cat. no. ab206386) was used according to the manufacturer's protocol.
Additionally, we performed immunohistochemistry (IHC) for the stem cell markers OLFM4 (mPCIS) and SOX9 (rPCIS), enterocyte marker Villin1 (mPCIS) and transit-amplifying cell marker KI67. After
Table 1
Composition of culture media.
Medium Base⁎ FBS R-Spondin-1
Noggin Wnt3a EGF N2/B27HEPES Species PCIS
WME WME Mouse and
rat
ADF⁎⁎ ADF Mouse and
rat
DF DF Mouse and
rat
WME-ENR WME + + + Mouse
WME-WENR WME + + + + Rat
ADF-ENR ADF + + + Mouse
ADF-WE ADF + + + Rat
ADF-WENR ADF + + + + Rat
DF-WENR DF + + + + Rat
OGM⁎⁎⁎ DF + + + Mouse and
rat
WME, Williams' Medium E; ADF, advanced Dulbecco's Modified Eagle Medium (DMEM)-F12; DF, DMEM-F12; OGM, organoid growing medium; EGF, epithe-lial growth factor; W, Wnt3a; E, EGF; N; Noggin, R; R-spondin-1.
⁎ All media were supplemented with Glutamax (final concentration 2 mM), D-Glucose (final concentration 25 mM), Gentamycin (1 μg/mL) and Fungizone (10 μL/mL).
⁎⁎ All ADF based media were supplemented with Sodium Pyruvate (1 mM). ⁎⁎⁎ OGM was kindly provided by Innovative Testing (INT) research group at Utrecht University of Applied Science. OGM contained DF as a base medium, supplemented with 10% FBS, Penicillin/Streptomycin, 10% (v/v) R-Spondin-1 conditioned medium, 10% (v/v) Wnt3a conditioned medium, DMH-1 (as an alternative to Noggin conditioned medium) and other undisclosed compounds.
deparaffinization, sections were rehydrated, and antigen retrieval was performed by incubating the sections in Tris/EDTA buffer (pH 9.0) for 15 min near the boiling point. Slides were incubated for 1 h with the following primary antibodies: anti-OLFM4 (1:200, Cell Signaling Technology PA, Leiden, The Netherlands; cat.no. 39141), anti-SOX9 (1:500, Thermo Fisher Scientific, Landsmeer, The Netherlands; cat.no. PA5-81966), anti-Villin (1:100, Proteintech, Manchester, UK; cat.no. 16488-1-AP), or anti-KI67 (1:750, Abcam, Cambridge, UK, cat.no. ab15580). Endogenous peroxidases were blocked by a 15 min incuba-tion in 0.1% H2O2in phosphate buffered saline. Then, sections were incubated with the secondary antibody (goat-anti-rabbit HRP; 1:50, Dako, Glostrup, Denmark; cat.no. P0448) for 30 min, and the sections stained for Villin were additionally incubated with a tertiary antibody (Rabbit-anti-goat HRP; 1:50, Dako, Glostrup, Denmark; cat.no. P0160) for 30 min. The antibodies were localized using the ImmPACT NovaRED kit (Vector Laboratories, Burlingame, USA, cat. no. SK-4805), followed by hematoxylin counterstaining. Images were obtained with a Nanozoomer Digital Pathology Scanner (NDP Scan U10074–01, Hamamatsu Photonics K.K., Japan). The TUNEL- and IHC-stained sec-tions were quantified using Aperio ImageScope software (Leica Biosystems Imaging, Inc., USA) as described previously (Ruigrok et al., 2019).
2.5. Enzyme-linked lectin assay (ELLA)
Enzyme-Linked Lectin Assay (ELLA) was performed to determine the mucus production by cultured PCIS. Medium samples were col-lected after each time point of incubation and stored at −20 °C. Maxisorp 96-wells microplates (ThermoFisher Scientific, Landsmeer, The Netherlands, cat. no. 442404) were coated for 2 h at 37 °C with 4 μg/mL wheat germ agglutinin (Sigma-Aldrich, Saint Louis, USA, cat.no. 101845324). The wells were blocked for 1 h at 37 °C with PBST. Subsequently, samples consisting of culture medium from cultured PCIS and standard curves of porcine mucin (Sigma-Aldrich, Saint Louis, USA, cat.no. 84082–64-4) in PBS/CaCl₂ (0.4 mM) in a range of 0.08–10 μg/ mL were added to the wells and incubated for 1 h at 37 °C. Next, the plate was incubated with 2 μg/mL horseradish peroxidase-labeled soybean agglutinin (Sigma-Aldrich, Saint Louis, USA, cat.no. L2650) for 1 h at 37 °C. After discarding the detection solution and a final washing, TMB substrate (Sigma, cat.no. 1002623873) was incubated for 5 min at ambient temperature in the dark. The reaction was stopped with 1 M H2SO4. Absorbance at A450was measured in a BMG FLuoStar-Omega. Signal-to-background ratio was calculated with a culture medium control.
2.6. RNA isolation and reverse-transcription polymerase chain reaction After incubation, 6 pooled PCIS were snap-frozen and stored at −80 °C until use. RNA was isolated using the FavorPrep™ Tissue Total RNA Mini Kit (Favorgen, Vienna, Austria), according to the manufac-turer's protocol. CDNA was prepared using the Reverse Transcription System (Promega, Leiden, The Netherlands) using a thermal cycler (10 min at 22 °C, 15 min at 42 °C and 5 min at 95 °C). Primers (Supplementary Table 2 and 3), and Fast Start Universal SYBR Green mastermix (Roche, Almere, The Netherlands) were used for real time quantitative PCR. RT-qPCR was performed with a ViiA7 real-time qPCR (Applied Biosystems, Bleiswijk, The Netherlands), using 1 cycle of 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C. MRNA expression was calculated as relative expression (2 -ΔCtmethod) compared to 0 h of incubation (timeline) or WME (media), using Gapdh as housekeeping gene.
2.7. Statistical analysis
All experiments were performed with at least three slices per animal (technical replicates) from a total of six to nine animals (biological
replicates). Statistical analyses were performed using GraphPad Prism 8.2 (GraphPad Software Inc., CA, USA). Treatment groups were com-pared using one-way ANOVA followed by Dunnett's multiple compar-isons test. A p-value < 0.05 was considered statistically significant. 3. Results
3.1. Mouse and rat PCIS contain all main epithelial cell subtypes before culture
To confirm that intestinal morphology and cellular heterogeneity is preserved in mouse and rat PCIS after the slicing procedure, we used histological and immunohistochemical analyses. Directly after PCIS preparation, main intestinal structures, such as villi and crypts, were well preserved and the epithelial monolayer appeared intact in all PCIS (Supplementary Fig. S1A and E). Enterocytes, cells that play an im-portant role in nutrient absorption and drug uptake, preserved their columnar shape and stayed polarized as shown by the apical expression of Villin (Fig. 1A). Mucin-producing goblet cells, recognized as polar-ized cells containing mucus-laden granules on the apical site, were also present directly after slicing (visualized by PAS stain;Fig. 1A and2A). Cells localized in the intestinal crypts, such as stem cells, transit-am-plifying (TA) cells and Paneth cells, are responsible for renewing the epithelial layer (Gehart and Clevers, 2019). In both mPCIS and rPCIS, stem cells and TA cells were present in the intestinal crypts directly after slicing as illustrated by the expression of stem cell markers OLFM4 (mPCIS) and SOX9 (rPCIS), and proliferating transit-amplifying (TA) cell marker KI67 (Fig. 1A and2A). Lastly, Paneth cells, recognized by the secretory eosinophilic vesicles, could be clearly detected in mPCIS and rPCIS directly after slice preparation (visualized by HE stain; Fig. 1A and2A). Taken together, we showed that both mouse and rat PCIS contained all main epithelial cell types, namely enterocytes, goblet cells, stem cells, TA cells and Paneth cells, prior to culturing. 3.2. Culturing of PCIS is associated with progressive damage of epithelium and loss of main epithelial cell subtypes
To investigate whether the different epithelial cell subtypes, nor-mally present in mPCIS and rPCIS, were affected during incubation in the standard culture medium, Williams' Medium E (WME), we carried out immunohistochemical/histological and gene expression analyses using cell-specific markers.
We observed clear signs of epithelial damage in mPCIS and rPCIS during incubation, which was morphologically manifested as disrupted monolayer with flattened epithelial cells (Supplementary Fig. S1D and H). Absorptive enterocytes, while still present after slice preparation, seemed to be lost early in culture. In mPCIS, protein expression of Villin was hardly detected after the start of incubation (Fig. 1A) and was significantly decreased during the whole culture period compared to 0 h (Fig. 1B). Moreover, mPCIS showed a decreased gene expression of enterocyte markers Vil1, Ezrin, Cdh1, and Alpi already at 24 h of in-cubation (Fig. 1F and Supplementary Fig. S2). In turn, rPCIS developed epithelial damage (Fig. 2B and Supplementary Fig. S1H) and showed reduced gene expression of Vil1, Ezrin, Cdh1, and Alpi (Fig. 2F and Supplementary Fig. S3) already after 3 h of incubation. We then mea-sured the expression of metabolizing enzymes, normally present in enterocytes. In line with previous studies (Bigaeva et al., 2019b;van de Kerkhof et al., 2005), the mRNA expression of Cyp1a1 and Cyp3a13 was decreased during incubation in mPCIS, (Fig. 1F, Supplementary Fig. S2), while expression of Cyp1a1 and Cyp3a62 was significantly down-regulated in rPCIS after 3 h of incubation (Fig. 2F and Supplementary Fig. S3). Overall, these results suggest that enterocytes suffer a sub-stantial damage during PCIS culture.
Concurrently, goblet cells disappeared during culture in both mPCIS and rPCIS (Fig. 1A and2A). Moreover, the production of mucus was decreased in mPCIS during culture (2.6 μg/mL after 24 h vs 0.55 μg/mL
after 120 h, (Fig. 1C). A similar trend was observed with mucus pro-duction by rPCIS, as it decreased, albeit not significantly, from 3.42 μg/ mL (normalized to 24 h) at 3 h to 0.56 μg/mL (normalized to 24 h) at 48 h of incubation (Fig. 2C). The gene expression of goblet cell marker Muc2 was significantly decreased in mPCIS, but not in rPCIS (Fig. 1F and 2F, Supplementary Figs. S2 and S3). Enteroendocrine cells also seemed to undergo changes during culture in both mPCIS and rPCIS, since all slices showed decreased mRNA expression of ChgA, and Sis (Fig. 1F and 2F, Supplementary Figs. S2 and S3). Of note, the en-teroendocrine cell marker Cldn4 (Nagatake et al., 2014) was sig-nificantly increased during mPCIS culture.
Next, we evaluated the impact of culturing on epithelial cell types found in the crypts – stem cells, TA cells and Paneth cells. Proper functioning and maintenance of intestinal stem cell niche is crucial for normal tissue homeostasis (Flanagan et al., 2018). In mPCIS, protein expression of stem cell marker OLFM4 dramatically dropped already at 24 h, reaching statistical significance at 48 h of incubation (Fig. 1A and E). Simultaneously, gene expression of Lgr5 and Olfm4 was significantly decreased at 24 h onwards (Fig. 1F and Supplementary Fig. S2). In rPCIS, the protein expression of SOX9 (Fig. 2A and E), a marker for active proliferating intestinal stem cells (Gonzalez et al., 2019), as well as mRNA expression of Lgr5, Olfm4 and Ascl (Fig. 2F and Supplemen-tary Fig. S3) were significantly decreased after 3 h of incubation.
A general feature of stem cells and TA cells is their proliferative capacity. Therefore, we measured protein and gene expression of KI67. Proliferating TA cells/stem cells completely disappeared in mPCIS after 72 h of incubation, illustrated by the diminished expression of KI67 protein (Fig. 1A and D). In rPCIS, KI67+cells were still partially present at 48 h, although KI67 levels showed significant decrease already at 3 h of incubation (Fig. 2A and D). Changes in KI67 protein expression were paralleled by decreased mRNA levels of Mki67 in both mPCIS and rPCIS during incubation. These results suggest that stem cells and TA cells are lost in PCIS shortly after the start of incubation. Culture-associated disruption of the stem cell niche was in accordance with the progressive loss of crypt structures (Supplementary Fig. S1D and H).
Together with stem cells and TA cells, Paneth cells also displayed signs of distress. In mPCIS, Paneth cells were detected only during first 48 h of incubation, while in rPCIS they completely disappeared already after 6 h (Fig. 1A andFig. 2A). Paneth cell markers Reg3b and Reg3g were significantly upregulated in mPCIS at 24 h, but downregulated at 120 h of incubation (Fig. 1F and Supplementary Fig. S2). In contrast, there was no change in Reg3b and Reg3g mRNA expression in rPCIS (Fig. 2F and Supplementary Fig. S3). Other markers typically expressed by Paneth cells – Ang4 and Lyz1 – were significantly decreased after 96 and 120 h of mPCIS incubation (Fig. 1F and supplementary Fig. S2). In rPCIS, Lyz2 was significantly decreased after 24 h of incubation (Fig. 2F and Supplementary Fig. S3).
Paneth cells protect and sustain stem cells by secreting bactericidal products and by providing essential niche signals. In particular, Wnt and Notch signaling play an important role in the maintenance of the intestinal crypt niche (Scoville et al., 2008). Therefore, we determined changes in the expression of genes associated with these two pathways in PCIS during culture. In mPCIS and rPCIS, Wnt target gene Axin2 was downregulated during culture, while Wnt effector Tcf4 and Notch li-gand Dll4 were significantly increased (Fig. 1F and 2F and Supple-mentary Fig. S2 and S3). The expression of Notch effector Hes1 re-mained unchanged in mPCIS and rPCIS during culture. However,
Olfm4, another target gene of Notch, was significantly downregulated during incubation. Furthermore, rPCIS showed significant down-regulation of Wnt target gene Ccnd1 (cyclin D1) already after 3 h of incubation; however, Wnt3a expression was not affected during culture. Overall, Wnt and Notch target genes are downregulated, indicating pathway inhibition.
In line with our earlier study (Bigaeva et al., 2019a), poor survival of epithelial cell subtypes was reflected by the decline in general via-bility of mPCIS and rPCIS during culture (Supplementary Fig. S1). A steady decrease in ATP content of mPCIS was concurrent with the histological deterioration during culture, which manifested by the de-velopment of edema, presence of necrotic and apoptotic cells, as well as by the loss of villi and crypts (Supplementary Fig. S1B and C). In turn, rPCIS showed an even more rapid loss of ATP content per slice and worsening of histological appearance during incubation (Supplemen-tary Fig. S1F and G).
Taken together, the culturing of PCIS induced progressive epithelial damage that manifested not only via general decrease in tissue viability, but also via the loss of main epithelial subtypes.
3.3. Culturing of PCIS induces intestinal cell death and triggers inflammatory and fibrogenic responses
To evaluate whether incubation of PCIS is associated with cell death of intestinal epithelial cells, we performed TUNEL staining (Fig. 3A). We observed an increase in DNA fragmentation, a marker of necrosis and apoptosis (Majtnerová and Roušar, 2018), in cultured PCIS com-pared to PCIS directly after slicing. However, quantitative analysis showed no significant changes in DNA fragmentation in mPCIS (Fig. 3B). In contrast, there was a significant, 2.5 fold, increase in TUNEL+staining in rPCIS after 48 h of incubation (Fig. 3C). Moreover, the pro-apoptotic gene Bax was significantly upregulated at 96 h in mPCIS (Fig. 3D and Supplementary Fig. S2) and at 48 h in rPCIS (Fig. 3D and Supplementary Fig. S3).
It has been shown that slice preparation and tissue handling trigger inflammatory signaling cascades and activate wound healing response (Bigaeva et al., 2019b). Therefore, we investigated the expression of genes related to inflammation and fibrosis in mPCIS and rPCIS (Sup-plementary Figs. S2 and S3). In all PCIS, inflammatory markers Il-6, Tnf, Il-1b, and Cxcl1 were significantly increased upon culture.
In mPCIS, the expression of fibrosis markers Col1a1 and Serpinh1 was not changed, while there was a significant decrease in the ex-pression of Acta2 and an increase in the exex-pression of Fn1, Tgfb1 and Mmp9 (Supplementary Fig. S2). In rPCIS, the expression of Col1a1 was not altered, expression of Acta2 was significantly decreased and ex-pression of Fn1, Serpinh1, Tgfb1 and Mmp9 was significantly increased (Supplementary Fig. S3). Overall, the expression of wound healing markers was in line with previous studies (Bigaeva et al., 2020; Iswandana et al., 2016b;Pham et al., 2015).
Taken together, we showed that in both mPCIS and rPCIS, loss of viability and epithelial damage were accompanied by increased in-testinal cell death and activated inflammatory and wound healing re-sponses.
Fig. 1. Effect of incubation time on epithelial cells in mouse precision-cut intestinal slices (mPCIS). Mouse PCIS were incubated in Williams' Medium E (WME) for 24,
48, 72, 96 and 120 h. (A) Representative images of immunohistochemistry, PAS and HE staining on paraffin sections; images were taken at 40× magnification, scale bars are 250 μm for IHC and PAS stainings or 50 μm for HE stainings. Arrows indicate paneth cells. Images were quantified using algorithmic analysis for positive pixels of (B) villin, (D) KI67 and (E) OLFM4. Bars show average positive pixel count ± SEM (n = 3–9). (C) Mucus concentration in the culture medium of mPCIS was determined using ELLA. Bars show mucus concentration over a period of 24 h ± SEM (n = 3–9). (F) Heatmap of the expression of epithelial cell type markers and Wnt signaling related genes in cultured mPCIS (n = 5–9). One-way ANOVA followed by Dunnett's post-test was used to calculate statistical differences compared to o hours of incubation (*p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.001). EEC, Enteroendocrine cells; TA, Transit-amplifying cells.
3.4. Presence of niche growth factors is beneficial for the survival of stem/ TA cells during culture of PCIS
The observed progressive loss of epithelial cells during culture calls for searching ways to ensure their survival ex vivo. We previously re-ported that the addition of stem cell niche growth factors, namely EGF, R-Spondin-1, Noggin and Wnt3a, to culture medium positively affects
ATP content and morphological appearance of mouse and rat PCIS (Bigaeva et al., 2019a). Wnt signaling promotes stem cell maintenance and proliferation. R-Spondin-1 acts as an agonist of Wnt signaling. EGF promotes proliferation, while Noggin inhibits BMP signaling and thereby differentiation of stem cells (Hong et al., 2017). In this study, we cultured mPCIS and rPCIS in media containing various combina-tions of EGF, Noggin, R-Spondin-1, Wnt3a, N2/B27 and HEPES
Fig. 2. Effect of incubation time on epithelial cells and rat precision-cut intestinal slices (rPCIS). Rat PCIS were incubated in Williams' Medium E (WME) for 3, 6, 24
and 48 h. (A) Representative images of immunohistochemistry, PAS and HE staining on paraffin sections; images were taken at 40× magnification, scale bars are 250 μm for IHC and PAS stainings or 50 μm for HE stainings. Arrows indicate paneth cells. (B) Histological score reflecting epithelial damage in rPCIS during culture. Paraffin sections were stained with hematoxylin-eosin and evaluated according to the histological score system described in methods (section 2.4). Each dot represents an epithelial damage score derived from PCIS of one rat. Data are expressed as mean ± SEM. (C) Mucus concentration in the culture medium of rPCIS was determined using ELLA. Bars show mucus concentration normalized to 24 h ± SEM (n = 3–6). Images were quantified using algorithmic analysis for positive pixels of (D) KI67 and (E) SOX9. Bars show average positive pixel count ± SEM, (n = 3–6). (F) Heatmap of the expression of epithelial cell type markers and Wnt signaling related genes in cultured rPCIS (n = 5–9). One-way ANOVA followed by Dunnett's post-test was used to calculate statistical differences compared to 0 h of incubation (**p ≤ 0.01; ***p ≤ 0.005; ****p ≤ 0.001). EEC, Enteroendocrine cells; TA, Transit-amplifying cells.
Fig. 3. Effect of incubation time on cell death in mouse and rat precision-cut intestinal slices (PCIS). MPCIS were incubated in Williams' Medium E (WME) for 24, 48,
72, 96 and 120 h, while rPCIS were incubated for 3, 6, 24 and 48 h in WME. (A) DNA strand breaks were visualized using TUNEL assay on paraffin sections; images were taken at 40× magnification, scale bars 250 μm. Images were quantified using algorithmic analysis for TUNEL positive pixels in (B) mPCIS and (C) rPCIS. Bars show average positive pixel count ( ± SEM), n = 3–9. (D) Heatmap of the expression of apoptosis, inflammation and fibrosis related genes in cultured PCIS (n = 5–9). One-way ANOVA followed by Dunnett's post-test was used to calculate statistical differences compared to 0 h of incubation (**p ≤ 0.01).
Fig. 4. Effect of different media and growth factors on
mouse precision-cut intestinal slice (mPCIS). Mouse PCIS were incubated for 96 h in Williams' Medium E (WME), Advanced DMEM-F12 (ADF) or DMEM-F12 (DF) with or without Epithelial Growth Factor (E), Noggin (N) and R-Spondin (R), or in Organoid (OGM) medium. (A) After in-cubation, viability of mPCIS was measured by the ATP (pmol) normalized for protein content (μg) in slices. (B) Absolute ATP values (pmol/slice). Data are expressed as mean ± SEM (n = 6). (C) HE stained sections were scored and dots show the histological score per individual mPCIS section, bars show mean ± SEM of the histological score. (D) Heatmap of the gene expression patterns of mPCIS. Algoritmic analysis of IHC stained sections for (E) Villin, (G) KI67, (H) OLFM4 and (I) TUNEL positive pixel count. Bars show positive pixels ± SEM (normalized to WME). (F) Mucus content in the media from mPCIS cultured in different media. Bars show mucus concentration ± SEM (μg/mL). One-way ANOVA and Dunnett's post-test were used to cal-culate statistical differences compared to WME (n = 5–6, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005, ****p ≤ 0.001). WME, Williams' Medium E; ADF, Advanced DMEM-F12 medium; DF, DMEM-F12 medium; ENR, epithelial growth factor (E), Noggin (N) and R-Spondin-1 (R); OGM, Organoid growing medium; EEC, Enteroendocrine cells; TA, Transit-amplifying cells.
(Table 1) and evaluated the survival of epithelial cell types in their presence. To this end, we used Williams' Medium E (WME), Dulbecco's Modified Eagle Medium (DMEM)/F-12 (DF) or Advanced DMEM/F-12 (ADF) as base medium, with or without growth factors EGF, Noggin and R-spondin-1 (ENR; for mPCIS), and additionally Wnt3a (WENR; for rPCIS). Tested media were selected based on pilot experiments, in which we screened 29 media of various compositions to determine which additives beneficially affect slice viability and cell proliferation (Supplementary Fig. S4). For instance, pilot experiments showed that the addition of FBS to PCIS cultures does not improve viability nor facilitate cell proliferation. Moreover, pilot data (Supplementary Fig. S4) confirmed that culture of mPCIS does not require the presence of Wnt3a in the culture medium, unlike rPCIS, which was first suggested by our proof-of-concept study (Bigaeva et al., 2019a). PCIS were also cultured in organoid-growing medium (OGM), which is produced and routinely used for growing 2D intestinal organoids in Research Centre for Healthy and Sustainable Living, Utrecht University of Applied Sci-ences (The Netherlands).
Fig. 4A and B show that culturing mPCIS in WME-ENR and ADF-ENR resulted in significantly higher ATP content at 96 h compared to slices cultured in WME or in the absence of the growth factors. Next, we employed a histological score system (Supplementary Table S1) to as-sess the morphological integrity of slices cultured in different media. The assigned histological score reflects the degree of epithelial damage (i.e. severity of epithelial cell necrosis and loss of cubic cell shape) as well as changes in overall mucosal architecture (i.e. loss of villi and crypts, and the extent of necrosis in the stroma). The lower the histo-logical score is (on a scale from 0 to 10), the more preserved structural integrity cultured PCIS display.Fig. 4C indicates that mPCIS cultured in WME-ENR, ADF-ENR as well as in ADF had lower histological score, although not significantly, compared to mPCIS cultured in WME. Si-milar to mPCIS, rPCIS tended to have slightly higher ATP content when cultured in the presence of growth factors for 24 h hours (Fig. 5A and B). However, none of the tested media could improve the histological score of cultured rPCIS as compared to WME (Fig. 5C). The observed beneficial effect of stem cell niche growth factors on general viability of mPCIS and rPCIS is in line with our earlier study (Bigaeva et al., 2019a). To answer the question whether the niche growth factors can rescue epithelial cells from culture-associated damage, we evaluated the ex-pression of epithelial cell subtype-specific markers in PCIS cultured in different media. Fig. 4D and Supplementary Fig. S5 summarize the transcriptional changes in mPCIS cultured for 96 h in modified media as compared to WME. Among the different epithelial cell types, only en-terocytes and stem/TA cells were significantly affected by the compo-sition of culture medium. In particular, ADF and DF induced mRNA expression of enterocyte markers Vil, Sis and Cyp3a13, although no changes in Villin protein expression were detected (Fig. 4E). This might be due to the fact that Villin is a marker for mature epithelial cells and not specifically for enterocytes (Sato et al., 2011). Trends towards in-creased (non-significantly) mRNA expression of goblet cell and Paneth cell markers were also observed in mPCIS cultured in ADF and DF. In contrast, the presence of niche growth factors made little difference for the survival of differentiated epithelial cell lineages in mPCIS at 96 h (Fig. 4D and E, Supplementary Fig. S5 and S6). Interestingly, mucus production was consistently reduced when mPCIS were cultured in medium containing niche factors (i.e., WME-ENR, ADF-ENR and OGM), as illustrated inFig. 4F. This is in line with mouse intestinal organoids in expansion medium containing niche factors (Tong et al., 2018;Yin et al., 2014), indicating that cells remain more in the undifferentiated state.
In contrast to differentiated epithelial cell types, undifferentiated stem/TA cells were impacted in mPCIS by the modified culture media, as shown by a significant upregulation of Mki67, Lgr5 and Olfm4 in WME-ENR, ADF-ENR or ADF alone (Fig. 4D and Supplementary Fig. S5). On a protein level, an elevated KI67 expression was observed in mPCIS cultured in these three media (Fig. 4G and Supplementary Fig.
S6), which is in agreement with the gene expression data. No significant changes were detected for OLFM4 protein expression, although it was 2-3-fold higher in all tested media compared to WME (Fig. 4H and Supplementary Fig. S6).
While WME-ENR, ADF and ADF-ENR had no impact on expression of genes related to Wnt and Notch signaling, apoptosis, inflammation or fibrosis, these genes were affected in mPCIS cultured for 96 h in OGM and especially DF (Fig. 4D and Supplementary Fig. S5). For instance, DF significantly downregulated Tcf4, Dll4, Tnf and Il-6, and OGM inhibited culture-induced expression of Mmp9. Lastly, none of the tested media were able to significantly diminish the DNA fragmentation compared to WME, as measured by TUNEL (Fig. 4I and Supplementary Fig. S6).
The effects of media with modified composition were less pro-nounced in rPCIS than those in mPCIS. As illustrated inFig. 5D, none of the tested media had a significant beneficial effect on the expression of gene markers specific for the differentiated epithelial cell types. Nevertheless, rPCIS cultured for 24 h in WME-WENR showed slightly higher mRNA expression of cell type-specific markers compared to WME, whereas rPCIS cultured in other media appeared to decrease it. There was also no difference in the scores that reflected only epithelial damage between the tested media (Fig. 5E). Similar to mPCIS, the ad-dition of niche growth factors to the medium resulted in a decreased mucus production (MUC2 by goblet cells, MUC1 and MUC4 by en-terocytes (Lindén et al., 2008) in rPCIS (Fig. 5F).
No significant changes in stem/TA cell markers were detected in rPCIS, except for those cultured in OGM that showed a marked decrease in Mki67, Olfm4 and Ascl2 expression (Fig. 5D and G, Supplementary Fig. S7 and S8). On the other hand, an elevated SOX9 protein expres-sion by WME-WENR might indicate that stem cells in rPCIS can be partially rescued when cultured in this medium (Fig. 5H and Supple-mentary Fig. S8). Interestingly, ADF medium supplemented with only EGF and Wnt3a was insufficient to support stem/TA cells, indicating that the presence of R-spondin-1 and/or Noggin is required for the synergetic effect of these four niche factors (Supplementary Figs. S9–S10).
The effects of modified media on the expression of apoptosis-, in-flammation- and fibrosis-related genes were minimal: only OGM sig-nificantly inhibited expression of Mmp9, similar to mPCIS (Fig. 5D). However, unlike in mPCIS, the addition of niche growth factors to the cultures of rPCIS significantly altered Wnt and Notch signaling by predominantly downregulating the Tcf4 gene. None of the tested media reduced DNA fragmentation in rPCIS at 24 h (Fig. 5I and Supplemen-tary Fig. S8).
Taken together, the effect of niche growth factors was limited to a partial rescue of stem/TA cell, but not other epithelial cell types, from culture-induced damage in PCIS. Furthermore, medium composition can impact Wnt and Notch signaling pathways that play a major role in intestinal homeostasis, as well as affect spontaneous onset of in-flammation and fibrosis in PCIS..
4. Discussion
Precision-cut intestinal slices (PCIS) are increasingly used to study intestinal (patho)physiology, drug efficacy, toxicity, transport and metabolism (Li et al., 2016). However, due to poor viability of PCIS during prolonged incubation, this ex vivo model is not used to its maximum potential. In this study, we provided evidence that freshly prepared mPCIS and rPCIS retain main epithelial cell types, namely absorptive enterocytes, goblet cells, stem cells, transit-amplifying (TA) cells and Paneth cells. We demonstrated that culturing of PCIS in the standard culture medium, Williams' Medium E (WME), induces the progressive epithelial damage, affecting the stem cell niche as well as secretory intestinal cell lineages. In particular, proliferative stem cells and TA cells, together with Paneth cells, were the most sensitive to culture-induced damage. Lastly, we showed that a portion of stem/TA cells could be rescued by the addition of niche growth factors to the
Fig. 5. Effect of different media and growth
fac-tors on rat precision-cut intestinal slice (rPCIS). Rat PCIS were incubated for 24 h in Williams' Medium E (WME), Advanced DMEM-F12 (ADF) or DMEM-F12 (DF) with or without Wnt3a (W), Epithelial Growth Factor (E), Noggin (N) and R-Spondin-1 (R), or in Organoid (OGM) medium. (A) After incubation, viability of rPCIS was mea-sured by the ATP (pmol) normalized for protein content (μg) in slices. (B) Absolute ATP values (pmol/slice). Data are expressed as mean ± SEM (n = 6). (C) HE stained sections were scored and dots show the total histological score per in-dividual rPCIS section, bars show mean ± SEM of the histological score. (E) Histological score reflecting epithelial damage in rPCIS during cul-ture. Each dot represents an epithelial damage score derived from PCIS of one rat. Data are ex-pressed as mean ( ± SEM). (D) Heatmap of the gene expression patterns of rPCIS. (F) Mucus content in the media from rPCIS cultured in
dif-ferent media. Bars show mucus
concentration ± SEM (μg/mL). Algoritmic ana-lysis of IHC stained sections for (G) KI67, (H) OLFM4 and (I) TUNEL positive pixel count. Bars show positive pixels ± SEM (normalized to WME). One-way ANOVA and Dunnett's post-test were used to calculate statistical differences compared to WME (n = 5–6, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005, ****p ≤ 0.001). WME, Williams' Medium E; ADF, Advanced DMEM-F12 medium; DF, DMEM-F12 medium; WENR, Wnt3a (W), epithelial growth factor (E), Noggin (N) and R-Spondin-1 (R); OGM, Organoid growing medium; EEC, Enteroendocrine cells; TA, Transit-amplifying cells.
culture medium, but it had no significant beneficial effect on other epithelial cell types.
Epithelial damage was shown by flattening of the epithelium and losing both epithelial polarization and differentiation markers. Moreover, we showed a progressive amount of cell death during in-cubation in WME. Both epithelial damage and cell death are likely to be induced by the mechanical trauma upon tissue collection and slice preparation. This causes disruption of the epithelial barrier and sub-sequently inflammatory response. Moreover, epithelial damage can be further induced by ischemia-reperfusion injury. Tissue handling as well as ischemia-reperfusion cause oxidative stress and induce an acute in-flammatory response (Bigaeva et al., 2019b; Roskott et al., 2010; Søfteland et al., 2019). By altering the way of handling and the pre-servation of the intestine before PCIS preparation, the epithelial da-mage might be partially prevented (Roskott et al., 2010). We showed that rPCIS were more prone to damage resulting in a decreased viability in comparison with mPCIS. This is in line with research of Søfteland et al., who found that rat intestines showed more tissue injury and in-flammatory cells and less cell-cell contact related protein expression – thus epithelial barrier function – upon cold storage compared to pig and human intestines (Søfteland et al., 2019).
In both mPCIS and rPCIS, we observed a dramatic loss of crypt-base cells – stem/TA cells and Paneth cells – early in the culture. One of the reasons might be a disrupted signaling between Paneth and stem cells, considering that Paneth cells secrete factors that are essential for maintaining the intestinal stem cell niche (Flanagan et al., 2018;Gehart and Clevers, 2019). Indeed, Wnt and Notch signaling pathways were affected in PCIS during culture, with concurrent decrease in mRNA and protein expression of stem/TA cell markers and in expression of Paneth-cell-secreted lysozyme (Lyz). On the other hand, we observed a change in Paneth cell morphology that indicated a more secretory cell lineage allocation. This might be associated with the increased secretion of Paneth-cell-specific antimicrobial peptides, since the expression of Reg3b and Reg3g was upregulated during the first 24 h of incubation in mPCIS. It has been shown that changed Paneth cell morphology is often seen during intestinal inflammation (Berkowitz et al., 2019;King et al., 2013), and that pro-inflammatory cytokines induce expression of anti-microbial peptides (Mukherjee and Hooper, 2015). In addition, the lack of a protective mucin layer may trigger compensatory production of antimicrobial peptides by Paneth cells in an attempt to restore mucosal defense barrier (Burger-van Paassen et al., 2012). Culture of mPCIS and rPCIS was indeed associated with a diminishing mucin production by goblet cells and enterocytes and a decreasing expression of Muc2 over time. However, further investigation, for example by measuring cyto-kine expression, is needed to elucidate the mechanism behind these observations.
Considering the rapid loss of crypt-base cells during incubation in WME, the renewal of the epithelial monolayer via proliferation and differentiation of stem/TA cells becomes insufficient to maintain epi-thelial integrity in PCIS. Indeed, we found that absorptive enterocytes, enteroendocrine cells and goblet cells are also lost during incubation of PCIS. The resulted compromised structural integrity of the intestinal epithelium is reflected in the general decrease in PCIS viability. Moreover, the loss of secretory cell lineages can be detrimental for the metabolic capacity of PCIS in culture. We previously showed that cul-ture is associated with the reduced enzymatic and metabolic activity in murine and human tissue slices, causing the inhibition of many path-ways related to the biosynthesis, endogenous metabolism and transport (Bigaeva et al., 2019b). The knowledge about alterations in tissue functionality and the survival of the cell types that provide it, is es-sential for the use of the PCIS model in toxicological and pharmacolo-gical studies.
Inclusion of the niche growth factors in the culture medium bene-fited PCIS viability and elevated the survival of stem/TA cells in in-testinal crypts, but had little or no effect on other epithelial cell lineages. A portion of stem/TA cells was rescued probably due to the
synergetic effect of R-spondin-1, Noggin and EGF in mPCIS, and ad-ditionally of Wnt3a in rPCIS, that supported stemness and cell pro-liferation. In mPCIS, other additives, such as N2 and B27, that contain protein usually found in serum, might enhance the PCIS viability by scavenging toxic radicals, and stimulating cell growth (Merker et al., 2016;Sato et al., 2011). The advantage of N2/B27 supplements was also shown in intestinal organoids and other stem cell cultures (Liu et al., 2006;Wachs et al., 2003). The impact of the modified culture media was more pronounced in mPCIS (incubated for 96 h) than in rPCIS (incubated for 24 h), suggesting a species-specific regulation of intestinal stem cell niche homeostasis. Wnt and Notch signaling are important in intestinal homeostasis; we showed that both pathways are inhibited during incubation in the standard medium (WME). One reason might be the acute injury and inflammation in PCIS caused by the slicing procedure. In a rat inflammatory bowel disease (IBD) model, similar expression patterns, i.e. inhibition of Wnt and Notch signaling genes, were determined upon induction of inflammation by TNBS (Xing et al., 2015). Other research showed that inhibition of one of the Notch pathways, could induce Wnt-signaling and Notch-ligand expression in mice (i.e. DLL4)(Tian et al., 2015). Only minor effects on Wnt and Notch-signaling genes were observed when PCIS were cultured in modified culture media. For example, Axin2, a Wnt target gene, was (partially) restored in mPCIS cultured in OGM (containing Wnt3a) and in rPCIS supplemented with niche growth factors and OGM (all con-taining Wnt3a). Of note, Wnt3a, R-Spondin-1 and Noggin were added to mPCIS and rPCIS cultures in a form of conditioned media derived from human cell lines producing mouse niche factors. Although mouse and rat proteins are nearly identical (Wnt3a 98.22%, R-Spondin1 96.56%, Noggin 100% sequence homology), there is no guarantee that these growth factors stimulate the same receptors with the same affinity in mice and rats. On the other hand, these same factors are also used for human organoid cultures, suggesting that they are sufficient in acti-vating the required WNT pathway that supports stem cell growth (Fujii et al., 2015).
In order to ensure the survival of all different epithelial cell types in PCIS during culture, not only the proliferation of stem/TA cells need to be improved, but also their differentiation to various secretory lineages. In this case, an additional supplementation of culture medium with factors that direct cell differentiation will be required. For instance, different combinations of compounds, such as CHIRR99021, DAPT, IWP-2 and Valproic acid have been shown to promote cell differentia-tion in intestinal organoids (Tong et al., 2018; Yin et al., 2014). Moreover, the use of small molecules like Y-27632, N-acetyl cysteine or nicotinamide, which are not directly related to the stem cell niche, also deserve further investigations (Kozuka et al., 2017;Yin et al., 2014). The ROCK inhibitor Y-27623 increases cell spreading and cell-cell and cell-matrix adhesion, thereby inhibiting anoikis, whereas N-acetyl cy-steine acts as an antioxidant and as a source for cycy-steine during the generation of glutathione. Nicotinamide is a precursor for NAD+and thereby can improve cell viability (Holmberg et al., 2017). Although any of the abovementioned molecules can be beneficial, we have to be cautious: such manipulations may alter physiological cell composition and/or cell type ratios of intestinal tissue in PCIS. Furthermore, these molecules might interact with the disease model by inhibiting in-flammation/fibrosis, for example in the case of Y-27632 (Holvoet et al., 2017).
An alternative strategy to improve PCIS viability and limit the epithelial damage is to optimize tissue handling and culturing in a way that would minimize ischemia-reperfusion injury or lower oxidative stress, among other factors. For instance, tissue injury upon ischemia-reperfusion can be prevented by supplementing preservation solutions during PCIS preparation with inflammatory, oxidant or anti-complement compounds (Mallick et al., 2004). In addition, injury by reoxygenation might be decreased by lowering the oxygen concentra-tion during incubaconcentra-tion (Ruigrok et al., 2019). Since intestinal epithe-lium, especially the part close to the villus tip, functions at
physiologically hypoxic conditions in vivo, this might be an important optimization step (Glover et al., 2016). While these strategies might benefit tissue viability, it is important to acknowledge that mechanic trauma, ischemia-reperfusion injury and oxidative stress – all con-tribute to the spontaneous onset of inflammation and fibrosis in PCIS. Therefore, if PCIS are used as a fibrosis model, then the modified conditions have to ensure not only tissue viability, but also the devel-opment of inflammatory and fibrogenic responses during culture.
In conclusion, we demonstrated epithelial cell heterogeneity in mPCIS and rPCIS, and characterized changes in cellular composition associated with the extensive epithelial damage driven by culturing. Moreover, we showed that a portion of crypt-base epithelial cells can be rescued by the addition of stem cell niche growth factors to the culture medium. Further optimization of PCIS cultures is needed to ensure both the extended life-span and the survival of epithelial cell types. With this study, we initiated the development of an improved PCIS model that would meet all the needs of pharmacological and toxicological studies. Supplementary data to this article can be found online athttps:// doi.org/10.1016/j.tiv.2020.104974.
Author contributions
E.B. and P.O. designed the study; C.B., E.B., M.H. and J.B. carried out experiments and analyzed the data; J.t.K. and S.V. developed and provided the organoid medium. C.B., E.B., M.H., and J.B. performed blind histomorphological scoring; K.v.S. performed the mucus mea-surements; M.T. and S.V. did the statistical analysis of the pilot ex-periments. C.B. and E.B. wrote the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ-ence the work reported in this paper.
Acknowledgements
We are thankful to Prof. C. Kuo and Prof. H. Clevers for providing the cell lines we used to produce R-spondin-1, Noggin and Wnt3a conditioned media. We also thank Prof. R. Bank for providing us with the cell culture facility. We also thank Prof. R. Coppes and M. Baanstra for helping with the Wnt3a assay. This study was supported by ZonMw (the Netherlands Organization for Health Research and Development), grant number 114025003. We also want to thank the GRIP (Groningen Research Institute for Pharmacy), for providing the PhD scholarship position.
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