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Progress on isolation and short-term ex-vivo culture of highly purified non-apoptotic human intestinal epithelial cells (IEC)
262 European Journal of Cell Biology 82, 262 ± 270 (2003, May) ¥ ¹ Urban & Fischer Verlag ¥ Jena http://www.urbanfischer.de/journals/ejcb
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Progress on isolation and short-term ex-vivo culture of highly purified non-apoptotic human intestinal epithelial cells (IEC) Johannes Grossmann1)a, Kathrin Walthera, Monika Artingera, Stephan Kiesslinga, Martin Steinkampb, WolfKuno Schmautzc, Florian Stadlerc, Frauke Batailled, Michael Schultza, J¸rgen Schˆlmericha, Gerhard Roglera a b c d
Department of Medicine I, University of Regensburg, Regensburg/Germany Department of Internal Medicine, University of Ulm, Ulm/Germany Department of Surgery, St Josef Hospital, Regensburg/Germany Institute of Pathology, University of Regensburg, Regensburg/Germany
Received November 7, 2002 Received in revised version January 10, 2003 Accepted January 15, 2003
Intestinal epithelial cells ± apoptosis ± anoikis ± cell culture ± cell anchorage Intestinal epithelial cells (IEC) form the largest surface of the human body and are of pivotal importance to digest and absorb nutrients. Furthermore these cells play a critical role shielding the organism against microorganisms and toxins present in the intestinal lumen. It is therefore not surprising that a large group of researchers take great interest in the study of these cells. However, to date it is a challenge to purify viable primary human intestinal epithelial cells and it has been even more fastidious to maintain IEC in culture ex-vivo as IEC undergo apoptosis within hours due to loss of cell anchorage (−anoikis×) following the isolation process. Over recent years the authors aimed to continuously improve the isolation technique for primary IEC, allowing a simple, effective and rapid isolation of highly purified non-apoptotic human IEC. In this study the newly improved method is presented and applied to establish ex-vivo cultures of highly purified, fully viable primary IEC displaying important functional properties, making these cells amenable for ex-vivo research on primary human intestinal epithelial cells.
Introduction Intestinal epithelial cells (IEC) of the small and large intestine play a key role for homeostasis of the organism. Depending on their localization in the intestinal tract they do not only resorb essentially all nutrients and fluids but furthermore they protect 1)
Dr. med. Johannes Grossmann, Department of Medicine I, University of Regensburg, D-93042 Regensburg/Germany, e-mail: [email protected], Fax: 49 941 944 7032.
the organism from an abundance of bacteria and toxins found even under normal conditions in the intestine. Furthermore, intestinal polyps leading to colon cancer ± one of the most common malignancies in the western world ± arise from transformation of colonic intestinal epithelial cells. It is therefore of prime interest to understand the physiology and pathophysiology of these cells, and not surprisingly a large community of scientists in research fields such as intestinal physiology, intestinal immunology and cancerogenesis finds interest to study these cells. The vast majority of this research, however, is not performed using so-called ™primary∫ IEC, meaning IEC derived directly (ex-vivo) from human intestinal tissue. This circumstance is in part due to lack of access to human tissue, in part though also due to difficulties to isolate, purify and cultivate these primary human IEC. Instead several commercially available IEC cell lines are being used, most of which are derived from transformed human tissue such as polyps and cancerous lesions (HT29, CaCo-2, T84). However, while these cells prove to be useful tools for studies on cell growth, proliferation and ion transport, findings derived from studies on cell lines not necessarily hold true when confirmation in primary human intestinal epithelial cells is undertaken (Daig et al., 2000; Panja et al., 1995). Therefore, also models of primary IEC cultures are needed and for many years, attempts now have been made to isolate these cells in order to generate ex-vivo primary IEC cultures (Kaeffer, 2002). Much of these studies were undertaken in rodents (Traber et al., 1991; Vidrich et al., 1988; Booth et al., 1995; Evans et al., 1994; Flint et al., 1991; Dzierzewicz et al., 2000) as for obviously better availability of tissue and independence from surgical schedules. Furthermore in rodents fetal tissue and intestine from suckling rodents can readily be obtained and successful primary cultures from these cells have been reported (Kondo et al., 1984; Evans et al., 1992; Brubaker
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and Vranic, 1987; Quaroni, 1985). These cells allow developmental studies but are unlikely to resemble the adult rodent IEC. Caution is warranted as findings obtained from studies on these cells need to be confirmed in human non-transformed IEC. Indeed, successful ex-vivo cultures of IEC from human fetal intestine confirm that primary IEC cultures can be obtained more easily from fetal tissue (Perreault and Beaulieu, 1998; Quaroni and Beaulieu, 1997; Chopra et al., 1987; Sanderson and Walker, 1995) ± yet it is probably even harder to get access to human fetal tissue for routine research use in most countries. Several protocols for isolation and ex-vivo culture of primary human IEC from surgical specimens or even endoscopic biopsies have been proposed in the literature (Aldhous et al., 2001; Pedersen et al., 2000; Deveney et al., 1996; Pang et al., 1996; Baten et al., 1992; Whitehead et al., 1999; Gibson et al., 1989; Panja, 2000; Fonti et al., 1994; Rogler et al., 1998; Cheng et al., 1984). These protocols differ in many aspects among each other such as the method chosen for detachment of IEC from the mucosa, the method chosen for purification of the IEC and the culture conditions chosen to maintain the IEC in culture. While each one of the protocols seems to have its own strengths, none of them has become a ™standard∫ in IEC research and the data presented have mostly not been able to conclusively demonstrate key points of a new ex-vivo culture method such as easy feasibility of the protocol, purity and viability of the cell preparation as well as functional integrity of the intestinal epithelial cells during ex-vivo culture. In this manuscript a protocol is presented which embraces our combined experience in isolating these cells over recent years and takes advantage of our studies on apoptosis induced by loss of cell anchorage (anoikis) in these cells (Frisch and Francis, 1994; Grossmann et al., 1998a, b, 2001; Rogler et al., 1998). Taking into account that IEC are highly anchorage dependent a novel method was developed, which isolates a highly purified population of IEC and provides the means to shorten the time of IEC detachment to 2 ± 3 minutes, making rapid readhesion of detached IEC possible, leading to survival of non-apoptotic IEC in ex-vivo culture. These cells display important functional properties which can be utilized in future studies on primary IEC.
Materials and methods Rapid isolation and purification of human intestinal epithelial cells Normal human intestinal mucosa from surgical specimens was obtained from patients undergoing surgery for large bowel neoplasia (> 10 cm distance to pathology). Following dissection of the mucosa into small strips (3 20 mm2) and mucus removal by 1 mM DTT (Sigma, Taufkirchen, Germany) in 50 ml Hanks× balanced salt solution (HBSS, PAA, Linz, Austria) (30 min at ambient temperature) mucosal strips are incubated in 1 mM EDTA (Sigma) for 10 minutes at 37 8C. Mucosal strips are briefly rinsed in HBSS (to eliminate already detached IEC), transferred to fresh HBSS at ambient temperature followed by 5 ± 10 vigorous shakes of the container. This procedure leads to instant detachment of IEC in a full-length crypt formation. After rapid removal of the mucosal strips (by passing the solution over a coarse mesh (400 mm, Rotilabo sieve, Carl Roth GmbH, Karlsruhe, Germany)) rapid purification of detached IEC is achieved by usage of a mesh filter (80 mm pore size, Sefar, Kansas City, Kansas) fixed with tape to a plastic ring (5 cm diameter, 2 cm height and 3 mm thickness). The suspension containing the IEC crypts is gently but rapidly passed over the mesh
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separating (™harvesting∫) the IEC crypts in the filter from single cells (erythrocytes, leukocytes, fibroblasts, etc.) which easily pass through the filter. The filter is then rapidly inverted, and purified intact IEC crypts are immediately washed out with culture medium (see below) at ambient temperature. This IEC crypt solution is then rapidly transferred to the ECM-coated (see below) culture dish (BD Falcon Multiwell tissue culture plate, 24-well flat bottom with lid, CatNo 353072, BD Labware, Heidelberg, Germany) for seeding of the cells. It is critical that the time interval from shaking of the container until seeding should be minimized. With little practice the entire procedure will not take longer than 2 ± 3 minutes.
Ex-vivo culture of primary human intestinal epithelial cells IEC crypts harvested in the mesh are washed out from the inverted filter by culture medium (Grossmann et al., 1998b) supplemented with glialderived neurotrophic factor (GDNF, 10 ng/ml, Upstate-Biozol, Eching, Germany). Not more than 5 ± 10 ml of culture medium should be used to wash (™harvest∫) the IEC from the inverted filter. The IEC crypt solution is then quickly transferred to tissue culture dishes coated with extracellular matrix (collagen I and collagen VI at 20 mg/m2 each, Sigma). To determine the best matrix for ex-vivo cultures of primary IEC the cells were seeded on the following ECM-components (each 20 mg/cm2): collagen I (CI), collagen VI (CVI) (Dako Diagnostika GmbH, Hamburg, Germany), laminin (LN), fibronectin (FN), collagen I plus IV, collagen I plus VI (all Sigma). Quantification of cell adhesion was determined by measurement of total DNA extracted only from adherent IEC after 12 hours of ex-vivo culture. IEC were seeded in 24well plates and little medium (250 ml/well) was used to seed the IEC crypts, enabling the cells to reach the matrix by sedimentation as fast as possible. After seeding, tissue culture plates are immediately transferred to an incubator (37 8C, 5% CO2) and left untouched for three hours. After three hours IEC have safely attached to the matrix and the remaining non-attached, apoptotic IEC are removed. After gently washing the wells with HBSS fresh culture medium is added and IEC can be used for further experiments.
DNA extraction and electrophoresis For DNA extraction IEC were harvested from the tissue culture plates at indicated time points. DNAwas extracted as described (Sambrook et al., 1989) and separated by 1.5% agarose gel electrophoresis. Gels were stained with propidium iodide and visualized by UV-light as described previously (Grossmann et al., 1998b).
Flow cytometry Flow cytometry was employed to assess IEC for 1) evidence of apoptosis, 2) cell purity during culture and 3) cell surface expression of Fas. Ad 1) Cells were harvested from the tissue culture plates at indicated time points by incubation in EDTA (1 mM) for 5 minutes followed by centrifugation at 2500 rpm for five minutes. Cells were resuspended in 70% ice-cold methanol and stored at 20 8C until analysis within 24 hours. Flow cytometry analysis to detect apoptotic cell populations was performed using propidium iodide stain according to standard technique (Darzynkiewicz et al., 1992) and as described previously (Grossmann et al., 2001). Ad 2) To ensure cell purity during ex-vivo culture, methanol-fixed adherent cells were harvested after 72 hours in culture and analyzed for cell-specific surface markers or cytosolic antigens by flow cytometry. Cells were washed twice in PBS followed by blocking (20 min PBS/2% FCS). Each batch of cells was incubated with isotype control antibodies (mouse IgG1-FITC, Coulter Immunotech Diagnostic, Hamburg, Germany), the epithelial surface glycoprotein 17 ± 1A marker Ber-EP-4 (Dako Diagnostika), the fibroblast marker anti-CD90/Thy-1 (AS02, Dianova, Hamburg, Germany) and the macrophage marker anti-CD68 (Dako Diagnostika). Ad 3) To demonstrate upregulation of Fas, ex-vivo cultivated IEC were harvested and methanol fixed. After washing and blocking (see above) cells were incubated with isotype control antibodies (IgG2, Sigma) and anti-Fas (Pharmingen-Becton Dickinson,
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Heidelberg, Germany). Per sample a minimum of 5 103 cells were measured and gating was applied on the sidescatter count to exclude debris and on the pulse width count to exclude remaining cell aggregates from analysis.
Western blot IEC cytosol was extracted at indicated times after initiation of ex-vivo culture or induction of FAS-induced apoptosis, and Western blot for caspase-3 and caspase-8 was performed as previously described (Grossmann et al., 1998b, 2001). Cytosol from IEC undergoing anoikis was used as positive control for caspase-3 and caspase-8 activation (Grossmann et al., 1998b, 2001).
MTT assay Metabolic activity of IEC in ex-vivo culture was assessed using the MTT assay according to the manufacturer×s instructions (Promega, Mannheim, Germany). For comparison of different IEC cultures, colorimetric values obtained by MTT assay (OD) were corrected for total DNA (ng/ ml) content per IEC sample tested. The HT-29 tumor epithelial cells (American Type Culture Association, Manassas, USA) served as a positive control. Total DNA was extracted and quantified using commercially available kits according to the manufacturer×s instructions (DNA mini-kit, Quiagen, Hilden, Germany, and Pico Green dsDNA Quantitation Kit, Molecular Probes, Leiden, The Netherlands).
Upregulation of Fas and induction of FASmediated apoptosis in primary IEC After 24 hours in culture IEC were incubated with IFN-g (20 and 40 ng/ ml, Roche, M¸nster, Germany) or medium. Following 12 hours of incubation with IFN-g or medium, FAS expression on IEC was analyzed by flow cytometry as described above, and IEC (following incubation with 20 ng/ml IFN-g) were treated with control medium or CH-11, a monoclonal antibody to Fas (100 ng/ml, Kamiya Biomedicals, Seattle, USA). After 120 minutes IEC cytosol was obtained and tested for activation of caspase-8 by Western blot as described above.
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demonstrates the critical steps of the new IEC isolation procedure. Using this technique the IEC yield per cm2 of mucosa could be markedly increased and microscopic evaluation of the IEC crypts as well as histological evaluation of the remaining mucosa demonstrated that intact additional full-length crypts were eluted from the mucosa with each shake (data not shown). In fact, the harvests from −shake and harvest× 2 ± 4 frequently contained most IEC crypts. However, if freshly isolated IEC are meant to be used for ex-vivo culture it is strongly recommended not to perform several shakes as anoikis will be induced in IEC awaiting the next −shake and harvest×. Instead the harvested IEC crypts should be transferred immediately to ECM-coated culture dishes for reattachment. To demonstrate that repeated isolation of non-apoptotic IEC can be performed ™safely∫ without IEC undergoing apoptosis in the mucosal tissue, IEC were isolated thirty, sixty and ninety minutes after the DTT incubation. IEC were analyzed for apoptosis using caspase-3-specific Western blot analysis, and freshly isolated IEC were kept in suspension to induce anoikis (Grossmann et al., 1998b) . As demonstrated in Figure 2 freshly detached IEC show no evidence of caspase-3 activation regardless of the time point when they were isolated from the mucosa. However ± as IEC are kept in suspension they will undergo anoikis as demonstrated by caspase-3 activation regardless of the time point of
Results Improvement of IEC isolation technique As primary human IEC separated from their underlying extracellular matrix during isolation will initiate anoikis within minutes of detachment, the primary goal of any isolation procedure for primary IEC needs to be minimization of the time interval between detachment from the mucosa and reattachment of IEC. Therefore, the previously reported onehour incubation of mucosal strips at 4 8C was substantially shortened to a 10-minute incubation at 37 8C. More importantly, following the incubation in EDTA the mucosal strips were once more carefully rinsed with HBSS in a course mesh (400 mm pore size) prior to shaking of the vessel. This step ensures that any IEC already detached even within these 10 minutes are removed. Therefore all IEC crypts harvested from the suspension after shaking of the mucosal strips are detached only seconds before. Subsequent rapid removal of the mucosal strips (400 mm mesh), filtration of the crypt suspension in the 80-mm filter, inversion of the filter and harvesting of purified crypts in culture medium can be performed within less than 2 minutes. To further increase the yield of purified IEC from a given piece of intestinal mucosa (which should measure at least 3 5 cm2) the incubation of the mucosal strips in fresh vials of EDTA (1 mM at 37 8C) can be repeated for at least four times. With each −shake and harvest× more freshly detached full-length IEC crypts become available for further experiments. Figure 1
Fig. 1. Schematic overview of the isolation protocol for primary human intestinal epithelial cells. Note that the mucosal strips (ms) can be reutilized for repeated incubations in EDTA to increase the yield of IEC crypts (* steps need to be performed very fast; AT ambient temperature).
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Fig. 2. Repeated isolation of non-apoptotic IEC from human mucosal tissue. In order to demonstrate that IEC remain non-apoptotic as long as they remain attached to the mucosa, caspase-3-specific Western blot analysis of repeatedly isolated IEC was performed. Following dissection, identical mucosal strips from human intestinal mucosa were utilized repeatedly (thirty (isolation 1), sixty (isolation 2) and ninety (isolation 3) minutes after the DTT incubation) to isolate and purify
intestinal epithelial cells. Essentialy all freshly isolated IEC (0 min after isolation) showed only inactive full-length caspase-3 (p32) regardless of the time point of isolation. When kept devoid of cell-matrix contact however IEC will undergo anoikis, regardless of the time point of isolation as demonstrated by activation and cleavage of caspase-3 yielding the 17 kDa fragment of activated caspase-3 (p17). Data representative of three experiments.
Fig. 3. Matrix reattachment and IEC survival. Isolated IEC crypts were seeded on different components of the extracellular matrix (C I: collagen 1; C VI: collagen VI; FN: fibronectin; LN: laminin). After two and four hours of ex-vivo culture attached (att) and non-attached (sus) IEC were harvested separately and assessed for caspase-3 activation by
Western blot. IEC kept on plastic (co) served as positive control. Attached IEC only contain inactive caspase-3 (p32) while IEC which did not attach and remained in suspension showed evidence of ongoing apoptosis as depicted by caspase-3 activation (p17). Data representative of five experiments.
isolation. These data demonstrate that IEC will remain nonapoptotic as long as they remain attached to the mucosa. IEC isolated at different time points following DTT incubation will initially be non-apoptotic and show no difference in susceptibility to anoikis. Repetition of the isolation procedure can be performed without loss of functionality within IEC.
collagen I plus VI was most effective in ensuring reattachment of primary IEC (data not shown). To assess the role of reattachment for survival floating and attached IEC were harvested separately after 2 and 4 hours of ex-vivo culture. As shown in Figure 3 attached IEC displayed no evidence of caspase-3 activation as only intact non-cleaved caspase-3 can be detected. However, IEC which were unable to reattach showed abundant caspase-3 activation as indicated by detection of the p17 subunit of caspase-3. These data demonstrate the pivotal role of cell-matrix reattachment for survival of primary human IEC. Attempts to increase IEC readhesion by generation of IEC single cells (by a 3-min incubation of the crypts in dispase (1.2 U/ml; Boehringer Mannheim, Mannheim, Germany)) did not increase survival or reattachment, presum-
Reattachment of ex-vivo isolated primary human IEC and survival Since IEC rapidly undergo anoikis following loss of cell anchorage, freshly isolated, non-apoptotic IEC were transferred to matrix-coated tissue culture dishes within 2 ± 3 minutes of detachment. Studies to determine the most suitable matrix for ex-vivo culture had shown that the combination of
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ably due to integrin and cell damage secondary to dispase incubation and prolongation of the time interval for IEC single cell isolation due to dispase incubation and mandatory washing of IEC following dispase incubation (data not shown).
Ex-vivo culture of primary human IEC ± morphology and purity Having demonstrated that IEC which did not reattach will undergo anoikis rather than survive non-adherent IEC were removed by rinsing of the tissue culture vessel after 3 hours of incubation. Subsequently medium was changed every 24 hours and IEC remained attached for 72 hours. Figure 4 displays a photograph of reattached, adherent epithelial cells in ex-vivo culture after 72 hours. Note the typical epithelial character of the flattened well adherent cells forming podocyte-like extrusions and in some instances little monolayers (arrow). However, after 72 hours IEC gradually show evidence of degeneration by shrinking of the cell body, retraction of cell podocytes and eventually detach as non-viable IEC. We have previously shown that cells, freshly isolated by our filter technique are > 95% intestinal epithelial cells (Grossmann et al., 2001). However, a main concern of ex-vivo culture of IEC remains contamination of the culture over time by other cell types ± in particular intestinal fibroblasts. To assess and ensure that the cells maintained in culture remain to be a pure population of IEC we analyzed the cell population in culture once more by flow cytometry after 72 hours. As shown in Figure 5 the cells kept in culture after 72 hours still represent a highly purified population of IEC whereas intestinal fibroblasts and macrophages account for a neglectably small population of cells (< 3%). Fig. 4. Morphology of ex-vivo cultured IEC. IEC were isolated as described above and kept in ex-vivo culture for 72 hours. Cells were studied for morphological features by phase-contrast microscopy (400 magnification). Note the flat spread out epithelial morphology of the cell bodies. Some cells even form small areas with cell aggregates of attached cells (arrow). However, some cells also show vacuolar degeneration at day three of ex-vivo culture, presumably indicating impending terminal differentiation.
Fig. 5. Purity of IEC maintained for 72 hours in ex-vivo culture. IEC were isolated as described above and kept in ex-vivo culture. After 72 hours in culture cells were analyzed by flow cytometry (FACS) to determine the type of adherent cells in culture. More than 90% of cells
Ex-vivo culture of primary human IEC ± viability and functionality Having demonstrated that the cells kept in ex-vivo culture are indeed purified IEC the next goal was to demonstrate viability and functionality of these cells. To assess ex-vivo cultured IEC for evidence of apoptosis cells were harvested at 24, 48 and 72 hours. Neither by DNA electrophoresis (Fig. 6A) nor by flow cytometry (FACS) (Fig. 6B) DNA fragmentation ± the hall-
stained positive for the epithelial cell marker whereas less than 3% of cells stained positive for the macrophage marker or the fibroblast marker AS02. Data representative for five ex-vivo cultures.
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Fig. 6. Ex-vivo cultivated IEC are non-apoptotic. Freshly isolated IEC were cultivated for 24, 48 and 72 hours as described above. A. DNA was extracted from ex-vivo cultured IEC at indicated time points and analyzed by electrophoresis for evidence of DNA fragmentation. IEC which had been in suspension culture for 180 minutes served as positive control (Co) (M 100-bp DNA marker). Ex-vivo cultivated human IEC show no evidence of DNA fragmentation. Data representative of five experiments. B. At indicated time points IEC were fixed and stained with propidium iodide for DNA content followed by flow cytometry. The data show that ex-vivo cultured IEC are non-apoptotic
as no −sub-G1× apoptotic cell population (arrow) could be detected. C. Ex-vivo cultivated IEC were lysed at indicated time points and activation of caspase-3 was assessed by Western blot. Freshly isolated IEC served as negative control (co 0) and apoptotic IEC (following 180 minutes suspension culture (Co sus)) served as positive control. In exvivo cultivated IEC only the inactive proform of caspase-3 (p32) can be detected, demonstrating that adherent IEC in culture are nonapoptotic. The activation of caspase-3, as depicted by appearance of the p17 subunit can be fully suppressed. Data representative of five experiments.
mark of apoptosis ± could be detected in ex-vivo cultured IEC. In accordance with these findings caspase-3-specific Western blot analysis (Fig. 6C) showed only full-length inactive caspase3 (p32) while the subunit (p17) of caspase-3 indicative of caspase-3 activation could not be detected. These data show that IEC remain non-apoptotic during ex-vivo culture. To demonstrate that IEC are not only non-apoptotic but show actual viability and metabolic activity the MTT assay ± a standard test assessing mitochondrial enzymatic activity ± was performed. As demonstrated in Figure 7 IEC show metabolic activity over the 72 hours of ex-vivo culture. Indeed primary IEC show significantly less metabolic activity (p < 0.05) than the control cells HT-29. This finding may well reflect the
difference between a transformed intestinal epithelial tumor cell and a physiological non-transformed intestinal epithelial cell. These data demonstrate that primary epithelial cells can be kept in a metabolically active state and remain viable during 72 hours of ex-vivo culture. To assess whether primary IEC kept in culture were not only vital but could be used for ex-vivo research further functional studies were performed. In reference to in-vitro findings obtained from studies on the cell line HT-29 (Abreu-Martin et al., 1995) the upregulation of the cell-surface receptor Fas in response to IFN-g incubation was tested in primary IEC. Following an overnight incubation with IFN-g a significant increase in Fas surface expression can be detected in ex-vivo cultivated IEC
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In summary, our data show that primary human IEC purified and cultivated according to the protocol presented are nonapoptotic, viable, metabolically active and suitable for functional studies involving cell metabolism and cytokine stimulation leading to expression of functional cell surface receptors.
Discussion
Fig. 7. Ex-vivo cultivated IEC and metabolic activity. Freshly isolated IEC were cultivated for 24, 48 and 72 hours as described above. IEC were assessed for metabolic activity using the MTT assay. The data show that IEC show persistent metabolic activity at indicated time points. Data show average standard deviation of five experiments. HT-29 tumor cells (HT-29) served as positive control and showed a significantly (* p < 0.05) higher metabolic activity when compared to primary IEC.
(Fig. 8A). To demonstrate that IFN-g incubation leads to upregulation of functional Fas, primary IEC were subsequently incubated with Fas-stimulatory antibody CH-11 to initiate Fas mediated apoptosis (Abreu-Martin et al., 1995). Ex-vivo cultivated IEC readily undergo Fas-mediated apoptosis following incubation with IFN-g and CH-11 as demonstrated by activation of caspase-8, a caspase known to be activated during Fasmediated apoptosis. Interestingly, primary IEC that were not preincubated with IFN-g showed no response to CH-11 (Fig. 8B).
Fig. 8. Ex-vivo cultivated IEC and functionality. IEC were cultivated ex-vivo as described above. A. After 24 hours IEC were incubated with IFN-g, and FAS expression on IEC was analyzed after 12 hours of incubation. Ex-vivo cultivated IEC show functional responsiveness to cytokine stimulation. B. Following IFN-g incubation (20 ng/ml) IEC were treated with control medium or Fas-stimulatory antibody CH-11. After 120 minutes IEC cytosol was obtained and tested for activation of
Apoptosis is a fundamental physiological mechanism inducing programmed death in distinct cells, thereby contributing just as importantly as proliferation to homeostasis of the organism. Colon intestinal epithelial cells proliferate in the stem cell region at the base of the intestinal crypt and subsequently migrate along the basement membrane to the intestinal lumen. As IEC reach the luminal surface, apoptosis is initiated and dying IEC are shed into the lumen as they undergo apoptosis (Potten and Allen, 1977; Gavrieli et al., 1992; Grossmann et al., 2002). The molecular mechanisms underlying the induction of IEC apoptosis in vivo are multifactorial with gradual loss of cell anchorage, a known trigger of apoptosis in various cell types (McGill et al., 1997; Re et al., 1994; Boudreau et al., 1995; Aoshiba et al., 1997) being most likely one factor contributing significantly to this physiological form of cell death at the end of the IEC life cycle (Probstmeier et al., 1990; Beaulieu, 1992; Koretz et al., 1991; Zutter and Santoro, 1990; Riedl et al., 1992). It was not before 1996 that Str‰ter et al. made the key observation that IEC isolation rapidly induces programmed cell death in these cells and furthermore demonstrated that loss of IEC anchorage to extracellular matrix by integrins was responsible for the induction of IEC apoptosis. Further studies could characterize the kinetics and molecular mechanisms
caspase-8 by Western blot. Ex-vivo cultivated IEC are functional as CH-11 incubation leads to activation of caspase-8 (as depicted by appearance of the p43, p41 and p18 subunits) while IEC incubated with medium show no evidence of caspase-8 (p48 and p45 isoforms of procaspase-8) activation. Data representative of three experiments. (Pos positive control, i.e. cytosol from IEC displaying caspase-8 activation)
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underlying IEC anoikis (Frisch and Francis, 1994; Grossmann et al., 1998b, 2001). IEC will initiate the activation of caspases ± key enzymes to initiate and execute apoptosis within a cell ± within minutes of loss of cell anchorage, leading to DNA fragmentation within few hours in essentially all IEC. Indeed, to our knowledge IEC are the most anchorage-dependent cells so far described in the human organism (Grossmann, 2002). Consequently, IEC isolation procedures involving longer incubation periods for enzymatic digestions, several centrifugation steps or even gradient centrifugation are bound to yield IEC undergoing apoptosis. IEC ± once detached ± will rapidly initiate anoikis. Based on our studies on IEC anoikis it became quite clear that the time interval between loss of cell anchorage and reattachment needed to be minimized to overcome induction of anoikis. While our manuscript published in 1998 involved a one-hour incubation in EDTA at 4 8C to inhibit caspase activation and centrifugation steps (Grossmann et al., 1998a) the protocol presented here involves only a ten-minute incubation in EDTA at 37 8C. After these ten minutes the mucosal strips still contain attached, though obviously ™loosened∫ IEC crypts which then are detached from the mucosa by shaking of the container. Following this maneuver the protocol takes advantage of the large crypt structure and quickly harvests all IEC crypts by a filter which can be everted immediately to yield highly purified IEC ready for immediate seeding in a tissue culture well. This rapid and very easy protocol indeed minimizes time of IEC detachment to 2 ± 3 minutes and ensures rapid reattachment of these highly anchorage-dependent cells. Aside from minimizing time for purification the 80-mm filter to harvest the crypts is of great value to ensure purity. Single cells such as fibroblasts, monocytes and lymphocytes will readily pass through the filter. However, the presence of mucus ± especially encountered with tissue samples from the sigmoid and rectum ± in the cell suspension following shaking of the container sometimes would be a problem ™clogging∫ the filter thereby hampering fast filtration and in particular purity. To solve this problem we found that reincubation of the mucosal strips in EDTA and repetition of the critical steps for detachment (see Figure 1) will not only substantially increase the overall yield of isolated IECs but also solves the ™mucus problem∫ as we experienced that only the first IEC harvest ± if at all ± will contain mucus in quantities sufficient to hamper filtration of the IEC. Therefore the repeated incubations in EDTA have substantially improved the IEC yield and solved the ™mucus problem∫ . To enable reattachment tissue culture plates need to be coated with ECM components and our studies confirmed the observation of others (Evans et al., 1994; Whitehead et al., 1987; Buset et al., 1987) that a collagen-based matrix is most efficient in allowing reattachment. As shown impressively by electron microscopy (Str‰ter et al., 1996) only IEC which have contact to the ECM and fully attach will survive while all other IEC will undergo apoptosis, i.e. anoikis. To eliminate dying cells from the culture we chose to wash the plates after three hours of ex-vivo culture as attached IEC by then have formed fully adherent flat cell aggregates while such IEC undergoing anoikis will have dissolved from the crypt and float in the medium. Therefore, after only three hours of ex-vivo culture a fully adherent viable population of purified IEC is available for in vitro studies. A main focus of this manuscript was to demonstrate viability, functionality and maintained purity of IEC during the course of the ex-vivo culture. As a consequence of the high purity of the
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isolate indeed IEC will not be overgrown by ± for instance fibroblasts ± during ex-vivo culture. Furthermore, the population of attached IEC will maintain viability and functionality in terms of responsiveness to cytokine stimulation and upregulation of functional cell-surface receptor expression. Unlike in previous reports, numerous non-subjective tests such as flow cytometry, DNA electrophoresis, Western blot, and MTT assay were employed to confirm this observation. Even though we found that the addition of the PI-3K stimulatory glial-derived neurotrophic factor (data not shown (Steinkamp et al., in press)) will improve survival of IEC during ex-vivo culture it was to date impossible to find conditions prolonging survival of substantial amounts of IEC beyond 3 days of ex-vivo culture. In summary, a further improved and simple method for isolation and short-term ex-vivo culture of viable primary human intestinal epithelial cells displaying important functional properties is presented, making human IEC from surgical specimens amenable for IEC research. Further studies are needed to assess different culture conditions to determine whether senescence of these cells after three days is due to genetic programming reflecting the physiological 3 ± 5-day life cycle of these cells or whether it is due to suboptimal conditions encountered during ex-vivo culture. Acknowledgements. This work is supported by grants ReForM A (J. Grossmann, M. Schultz) and ReForM B (J. Grossmann) grants (Medical Research Funding Program of the University of Regensburg, Regensburg, Germany) as well grants GR1523/3 ± 1 (J. Grossmann) and SFB585 (J. Grossmann, F. Bataille, G. Rogler) from the Deutsche Forschungsgemeinschaft, Bonn, Germany. We wish to thank the Department of Surgery and the Institute of Pathology at the University of Regensburg and the Department of Surgery, St. Josefs Hospital, Regensburg for providing surgical specimens.
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Pang, G., Buret, A., O×Loughlin, E., Smith, A., Batey, R., Clancy, R. (1996): Immunologic, functional, and morphological characterization of three new human small intestinal epithelial cell lines. Gastroenterology 111, 8 ± 18. Panja, A. (2000): A novel method for the establishment of a pure population of nontransformed human intestinal primary epithelial cell (HIPEC) lines in long term culture. Lab. Invest. 80, 1473 ± 1475. Panja, A., Siden, E., Mayer, L. (1995): Synthesis and regulation of accessory/proinflammatory cytokines by intestinal epithelial cells. Clin. Exp. Immunol. 100, 298 ± 305. Pedersen, G., Saermark, T., Giese, B., Hansen, A., Drag, B., Brynskov, J. (2000): A simple method to establish short-term cultures of normal human colonic epithelial cells from endoscopic biopsy specimens. Comparison of isolation methods, assessment of viability and metabolic activity. Scand. J. Gastroenterol. 35, 772 ± 780. Perreault, N., and Beaulieu, J. F. (1998): Primary cultures of fully differentiated and pure human intestinal epithelial cells. Exp. Cell Res. 245, 34 ± 42. Potten, C. S., and Allen, T. D. (1977): Ultrastructure of cell loss in intestinal mucosa. J. Ultrastruct. Res. 60, 272 ±277. Probstmeier, R., Martini, R., Schachner, M. (1990): Expression of J1/ tenascin in the crypt-villus unit of adult mouse small intestine: implications for its role in epithelial cell shedding. Development 109, 313 ± 321. Quaroni, A. (1985): Development of fetal rat intestine in organ and monolayer culture. J. Cell Biol. 100, 1611 ± 1622. Quaroni, A., and Beaulieu, J. F. (1997): Cell dynamics and differentiation of conditionally immortalized human intestinal epithelial cells. Gastroenterology 113, 1198 ± 1213. Re, F., Zanetti, A., Sironi, M., Polentarutti, N., Lanfrancone, L., Dejana, E., Colotta, F. (1994): Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells. J. Cell Biol. 127, 537 ± 546. Riedl, S. E., Faissner, A., Schlag, P., Von Herbay, A., Koretz, K., Moller, P. (1992): Altered content and distribution of tenascin in colitis, colon adenoma, and colorectal carcinoma. Gastroenterology 103, 400 ± 406. Rogler, G., Daig, R., Aschenbrenner, E., Vogl, D., Falk, W., Gross, V., Schˆlmerich, J., Andus, T. (1998): Long term primary cultures of human intestinal epithelial cells. Lab. Invest. 78, 889 ± 890. Sambrook, J., Fritsch, E. F., Maniatis, T. 1989. Commonly used techniques in molecular cloning. In: Molecular cloning. Cold Spring Harbor Laboratory Press, New York. E3 ± E11. Sanderson, I. R., and Walker, W. A. (1995): Establishment of a human fetal small intestinal epithelial cell line. Int. Arch. Allergy Immunol. 107, 396 ± 397. Steinkamp, M., Geerling, I., Seufferlein, T., Egger, B., Reimann, J., von Boyen, G., Grossmann, J., Adler, G., Reinshagen, M. (2003): Glial derived neurotrophic factor is upregulated in IBD and inhibits apoptosis in colon epithelial cells. Gastroenterology, in press. Str‰ter, J., Wedding, U., Barth, T. F. E., Koretz, K., Elsing, C., Moller, P. (1996): Rapid onset of apoptosis in vitro follows disruption of b1integrin/matrix interactions in human colonic crypt cells. Gastroenterology 110, 1776 ± 1784. Traber, P. G., Gumucio, D. L., Wang, W. (1991): Isolation of intestinal epithelial cells for the study of differential gene expression along the crypt-villus axis. Am. J. Physiol. 260, G895 ± 903. Vidrich, A., Ravindranath, R., Farsi, K., Targan, S. (1988): A method for the rapid establishment of normal adult mammalian colonic epithelial cell cultures. In Vitro Cell Dev. Biol. 24, 188 ± 194. Whitehead, R. H., Brown, A., Bhathal, P. S. (1987): A method for the isolation and culture of human colonic crypts in collagen gels. In vitro 23, 436 ± 442. Whitehead, R. H., Demmler, K., Rockman, S. P., Watson, N. K. (1999): Clonogenic growth of epithelial cells from normal colonic mucosa from both mice and humans. Gastroenterology 117, 858 ± 865. Zutter, M. M., and Santoro, S. A. (1990): Widespread histologic distribution of the alpha 2 beta 1 integrin cell-surface collagen receptor. Am. J. Pathol. 137, 113 ± 120.
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Progress on isolation and short-term ex-vivo culture of highly purified non-apoptotic human intestinal epithelial cells (IEC) Johannes Grossmann1)a, Kathrin Walthera, Monika Artingera, Stephan Kiesslinga, Martin Steinkampb, WolfKuno Schmautzc, Florian Stadlerc, Frauke Batailled, Michael Schultza, J¸rgen Schˆlmericha, Gerhard Roglera a b c d
Department of Medicine I, University of Regensburg, Regensburg/Germany Department of Internal Medicine, University of Ulm, Ulm/Germany Department of Surgery, St Josef Hospital, Regensburg/Germany Institute of Pathology, University of Regensburg, Regensburg/Germany
Received November 7, 2002 Received in revised version January 10, 2003 Accepted January 15, 2003
Intestinal epithelial cells ± apoptosis ± anoikis ± cell culture ± cell anchorage Intestinal epithelial cells (IEC) form the largest surface of the human body and are of pivotal importance to digest and absorb nutrients. Furthermore these cells play a critical role shielding the organism against microorganisms and toxins present in the intestinal lumen. It is therefore not surprising that a large group of researchers take great interest in the study of these cells. However, to date it is a challenge to purify viable primary human intestinal epithelial cells and it has been even more fastidious to maintain IEC in culture ex-vivo as IEC undergo apoptosis within hours due to loss of cell anchorage (−anoikis×) following the isolation process. Over recent years the authors aimed to continuously improve the isolation technique for primary IEC, allowing a simple, effective and rapid isolation of highly purified non-apoptotic human IEC. In this study the newly improved method is presented and applied to establish ex-vivo cultures of highly purified, fully viable primary IEC displaying important functional properties, making these cells amenable for ex-vivo research on primary human intestinal epithelial cells.
Introduction Intestinal epithelial cells (IEC) of the small and large intestine play a key role for homeostasis of the organism. Depending on their localization in the intestinal tract they do not only resorb essentially all nutrients and fluids but furthermore they protect 1)
Dr. med. Johannes Grossmann, Department of Medicine I, University of Regensburg, D-93042 Regensburg/Germany, e-mail: [email protected], Fax: 49 941 944 7032.
the organism from an abundance of bacteria and toxins found even under normal conditions in the intestine. Furthermore, intestinal polyps leading to colon cancer ± one of the most common malignancies in the western world ± arise from transformation of colonic intestinal epithelial cells. It is therefore of prime interest to understand the physiology and pathophysiology of these cells, and not surprisingly a large community of scientists in research fields such as intestinal physiology, intestinal immunology and cancerogenesis finds interest to study these cells. The vast majority of this research, however, is not performed using so-called ™primary∫ IEC, meaning IEC derived directly (ex-vivo) from human intestinal tissue. This circumstance is in part due to lack of access to human tissue, in part though also due to difficulties to isolate, purify and cultivate these primary human IEC. Instead several commercially available IEC cell lines are being used, most of which are derived from transformed human tissue such as polyps and cancerous lesions (HT29, CaCo-2, T84). However, while these cells prove to be useful tools for studies on cell growth, proliferation and ion transport, findings derived from studies on cell lines not necessarily hold true when confirmation in primary human intestinal epithelial cells is undertaken (Daig et al., 2000; Panja et al., 1995). Therefore, also models of primary IEC cultures are needed and for many years, attempts now have been made to isolate these cells in order to generate ex-vivo primary IEC cultures (Kaeffer, 2002). Much of these studies were undertaken in rodents (Traber et al., 1991; Vidrich et al., 1988; Booth et al., 1995; Evans et al., 1994; Flint et al., 1991; Dzierzewicz et al., 2000) as for obviously better availability of tissue and independence from surgical schedules. Furthermore in rodents fetal tissue and intestine from suckling rodents can readily be obtained and successful primary cultures from these cells have been reported (Kondo et al., 1984; Evans et al., 1992; Brubaker
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and Vranic, 1987; Quaroni, 1985). These cells allow developmental studies but are unlikely to resemble the adult rodent IEC. Caution is warranted as findings obtained from studies on these cells need to be confirmed in human non-transformed IEC. Indeed, successful ex-vivo cultures of IEC from human fetal intestine confirm that primary IEC cultures can be obtained more easily from fetal tissue (Perreault and Beaulieu, 1998; Quaroni and Beaulieu, 1997; Chopra et al., 1987; Sanderson and Walker, 1995) ± yet it is probably even harder to get access to human fetal tissue for routine research use in most countries. Several protocols for isolation and ex-vivo culture of primary human IEC from surgical specimens or even endoscopic biopsies have been proposed in the literature (Aldhous et al., 2001; Pedersen et al., 2000; Deveney et al., 1996; Pang et al., 1996; Baten et al., 1992; Whitehead et al., 1999; Gibson et al., 1989; Panja, 2000; Fonti et al., 1994; Rogler et al., 1998; Cheng et al., 1984). These protocols differ in many aspects among each other such as the method chosen for detachment of IEC from the mucosa, the method chosen for purification of the IEC and the culture conditions chosen to maintain the IEC in culture. While each one of the protocols seems to have its own strengths, none of them has become a ™standard∫ in IEC research and the data presented have mostly not been able to conclusively demonstrate key points of a new ex-vivo culture method such as easy feasibility of the protocol, purity and viability of the cell preparation as well as functional integrity of the intestinal epithelial cells during ex-vivo culture. In this manuscript a protocol is presented which embraces our combined experience in isolating these cells over recent years and takes advantage of our studies on apoptosis induced by loss of cell anchorage (anoikis) in these cells (Frisch and Francis, 1994; Grossmann et al., 1998a, b, 2001; Rogler et al., 1998). Taking into account that IEC are highly anchorage dependent a novel method was developed, which isolates a highly purified population of IEC and provides the means to shorten the time of IEC detachment to 2 ± 3 minutes, making rapid readhesion of detached IEC possible, leading to survival of non-apoptotic IEC in ex-vivo culture. These cells display important functional properties which can be utilized in future studies on primary IEC.
Materials and methods Rapid isolation and purification of human intestinal epithelial cells Normal human intestinal mucosa from surgical specimens was obtained from patients undergoing surgery for large bowel neoplasia (> 10 cm distance to pathology). Following dissection of the mucosa into small strips (3 20 mm2) and mucus removal by 1 mM DTT (Sigma, Taufkirchen, Germany) in 50 ml Hanks× balanced salt solution (HBSS, PAA, Linz, Austria) (30 min at ambient temperature) mucosal strips are incubated in 1 mM EDTA (Sigma) for 10 minutes at 37 8C. Mucosal strips are briefly rinsed in HBSS (to eliminate already detached IEC), transferred to fresh HBSS at ambient temperature followed by 5 ± 10 vigorous shakes of the container. This procedure leads to instant detachment of IEC in a full-length crypt formation. After rapid removal of the mucosal strips (by passing the solution over a coarse mesh (400 mm, Rotilabo sieve, Carl Roth GmbH, Karlsruhe, Germany)) rapid purification of detached IEC is achieved by usage of a mesh filter (80 mm pore size, Sefar, Kansas City, Kansas) fixed with tape to a plastic ring (5 cm diameter, 2 cm height and 3 mm thickness). The suspension containing the IEC crypts is gently but rapidly passed over the mesh
Primary human IEC isolation and culture 263
separating (™harvesting∫) the IEC crypts in the filter from single cells (erythrocytes, leukocytes, fibroblasts, etc.) which easily pass through the filter. The filter is then rapidly inverted, and purified intact IEC crypts are immediately washed out with culture medium (see below) at ambient temperature. This IEC crypt solution is then rapidly transferred to the ECM-coated (see below) culture dish (BD Falcon Multiwell tissue culture plate, 24-well flat bottom with lid, CatNo 353072, BD Labware, Heidelberg, Germany) for seeding of the cells. It is critical that the time interval from shaking of the container until seeding should be minimized. With little practice the entire procedure will not take longer than 2 ± 3 minutes.
Ex-vivo culture of primary human intestinal epithelial cells IEC crypts harvested in the mesh are washed out from the inverted filter by culture medium (Grossmann et al., 1998b) supplemented with glialderived neurotrophic factor (GDNF, 10 ng/ml, Upstate-Biozol, Eching, Germany). Not more than 5 ± 10 ml of culture medium should be used to wash (™harvest∫) the IEC from the inverted filter. The IEC crypt solution is then quickly transferred to tissue culture dishes coated with extracellular matrix (collagen I and collagen VI at 20 mg/m2 each, Sigma). To determine the best matrix for ex-vivo cultures of primary IEC the cells were seeded on the following ECM-components (each 20 mg/cm2): collagen I (CI), collagen VI (CVI) (Dako Diagnostika GmbH, Hamburg, Germany), laminin (LN), fibronectin (FN), collagen I plus IV, collagen I plus VI (all Sigma). Quantification of cell adhesion was determined by measurement of total DNA extracted only from adherent IEC after 12 hours of ex-vivo culture. IEC were seeded in 24well plates and little medium (250 ml/well) was used to seed the IEC crypts, enabling the cells to reach the matrix by sedimentation as fast as possible. After seeding, tissue culture plates are immediately transferred to an incubator (37 8C, 5% CO2) and left untouched for three hours. After three hours IEC have safely attached to the matrix and the remaining non-attached, apoptotic IEC are removed. After gently washing the wells with HBSS fresh culture medium is added and IEC can be used for further experiments.
DNA extraction and electrophoresis For DNA extraction IEC were harvested from the tissue culture plates at indicated time points. DNAwas extracted as described (Sambrook et al., 1989) and separated by 1.5% agarose gel electrophoresis. Gels were stained with propidium iodide and visualized by UV-light as described previously (Grossmann et al., 1998b).
Flow cytometry Flow cytometry was employed to assess IEC for 1) evidence of apoptosis, 2) cell purity during culture and 3) cell surface expression of Fas. Ad 1) Cells were harvested from the tissue culture plates at indicated time points by incubation in EDTA (1 mM) for 5 minutes followed by centrifugation at 2500 rpm for five minutes. Cells were resuspended in 70% ice-cold methanol and stored at 20 8C until analysis within 24 hours. Flow cytometry analysis to detect apoptotic cell populations was performed using propidium iodide stain according to standard technique (Darzynkiewicz et al., 1992) and as described previously (Grossmann et al., 2001). Ad 2) To ensure cell purity during ex-vivo culture, methanol-fixed adherent cells were harvested after 72 hours in culture and analyzed for cell-specific surface markers or cytosolic antigens by flow cytometry. Cells were washed twice in PBS followed by blocking (20 min PBS/2% FCS). Each batch of cells was incubated with isotype control antibodies (mouse IgG1-FITC, Coulter Immunotech Diagnostic, Hamburg, Germany), the epithelial surface glycoprotein 17 ± 1A marker Ber-EP-4 (Dako Diagnostika), the fibroblast marker anti-CD90/Thy-1 (AS02, Dianova, Hamburg, Germany) and the macrophage marker anti-CD68 (Dako Diagnostika). Ad 3) To demonstrate upregulation of Fas, ex-vivo cultivated IEC were harvested and methanol fixed. After washing and blocking (see above) cells were incubated with isotype control antibodies (IgG2, Sigma) and anti-Fas (Pharmingen-Becton Dickinson,
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Heidelberg, Germany). Per sample a minimum of 5 103 cells were measured and gating was applied on the sidescatter count to exclude debris and on the pulse width count to exclude remaining cell aggregates from analysis.
Western blot IEC cytosol was extracted at indicated times after initiation of ex-vivo culture or induction of FAS-induced apoptosis, and Western blot for caspase-3 and caspase-8 was performed as previously described (Grossmann et al., 1998b, 2001). Cytosol from IEC undergoing anoikis was used as positive control for caspase-3 and caspase-8 activation (Grossmann et al., 1998b, 2001).
MTT assay Metabolic activity of IEC in ex-vivo culture was assessed using the MTT assay according to the manufacturer×s instructions (Promega, Mannheim, Germany). For comparison of different IEC cultures, colorimetric values obtained by MTT assay (OD) were corrected for total DNA (ng/ ml) content per IEC sample tested. The HT-29 tumor epithelial cells (American Type Culture Association, Manassas, USA) served as a positive control. Total DNA was extracted and quantified using commercially available kits according to the manufacturer×s instructions (DNA mini-kit, Quiagen, Hilden, Germany, and Pico Green dsDNA Quantitation Kit, Molecular Probes, Leiden, The Netherlands).
Upregulation of Fas and induction of FASmediated apoptosis in primary IEC After 24 hours in culture IEC were incubated with IFN-g (20 and 40 ng/ ml, Roche, M¸nster, Germany) or medium. Following 12 hours of incubation with IFN-g or medium, FAS expression on IEC was analyzed by flow cytometry as described above, and IEC (following incubation with 20 ng/ml IFN-g) were treated with control medium or CH-11, a monoclonal antibody to Fas (100 ng/ml, Kamiya Biomedicals, Seattle, USA). After 120 minutes IEC cytosol was obtained and tested for activation of caspase-8 by Western blot as described above.
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demonstrates the critical steps of the new IEC isolation procedure. Using this technique the IEC yield per cm2 of mucosa could be markedly increased and microscopic evaluation of the IEC crypts as well as histological evaluation of the remaining mucosa demonstrated that intact additional full-length crypts were eluted from the mucosa with each shake (data not shown). In fact, the harvests from −shake and harvest× 2 ± 4 frequently contained most IEC crypts. However, if freshly isolated IEC are meant to be used for ex-vivo culture it is strongly recommended not to perform several shakes as anoikis will be induced in IEC awaiting the next −shake and harvest×. Instead the harvested IEC crypts should be transferred immediately to ECM-coated culture dishes for reattachment. To demonstrate that repeated isolation of non-apoptotic IEC can be performed ™safely∫ without IEC undergoing apoptosis in the mucosal tissue, IEC were isolated thirty, sixty and ninety minutes after the DTT incubation. IEC were analyzed for apoptosis using caspase-3-specific Western blot analysis, and freshly isolated IEC were kept in suspension to induce anoikis (Grossmann et al., 1998b) . As demonstrated in Figure 2 freshly detached IEC show no evidence of caspase-3 activation regardless of the time point when they were isolated from the mucosa. However ± as IEC are kept in suspension they will undergo anoikis as demonstrated by caspase-3 activation regardless of the time point of
Results Improvement of IEC isolation technique As primary human IEC separated from their underlying extracellular matrix during isolation will initiate anoikis within minutes of detachment, the primary goal of any isolation procedure for primary IEC needs to be minimization of the time interval between detachment from the mucosa and reattachment of IEC. Therefore, the previously reported onehour incubation of mucosal strips at 4 8C was substantially shortened to a 10-minute incubation at 37 8C. More importantly, following the incubation in EDTA the mucosal strips were once more carefully rinsed with HBSS in a course mesh (400 mm pore size) prior to shaking of the vessel. This step ensures that any IEC already detached even within these 10 minutes are removed. Therefore all IEC crypts harvested from the suspension after shaking of the mucosal strips are detached only seconds before. Subsequent rapid removal of the mucosal strips (400 mm mesh), filtration of the crypt suspension in the 80-mm filter, inversion of the filter and harvesting of purified crypts in culture medium can be performed within less than 2 minutes. To further increase the yield of purified IEC from a given piece of intestinal mucosa (which should measure at least 3 5 cm2) the incubation of the mucosal strips in fresh vials of EDTA (1 mM at 37 8C) can be repeated for at least four times. With each −shake and harvest× more freshly detached full-length IEC crypts become available for further experiments. Figure 1
Fig. 1. Schematic overview of the isolation protocol for primary human intestinal epithelial cells. Note that the mucosal strips (ms) can be reutilized for repeated incubations in EDTA to increase the yield of IEC crypts (* steps need to be performed very fast; AT ambient temperature).
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Primary human IEC isolation and culture 265
Fig. 2. Repeated isolation of non-apoptotic IEC from human mucosal tissue. In order to demonstrate that IEC remain non-apoptotic as long as they remain attached to the mucosa, caspase-3-specific Western blot analysis of repeatedly isolated IEC was performed. Following dissection, identical mucosal strips from human intestinal mucosa were utilized repeatedly (thirty (isolation 1), sixty (isolation 2) and ninety (isolation 3) minutes after the DTT incubation) to isolate and purify
intestinal epithelial cells. Essentialy all freshly isolated IEC (0 min after isolation) showed only inactive full-length caspase-3 (p32) regardless of the time point of isolation. When kept devoid of cell-matrix contact however IEC will undergo anoikis, regardless of the time point of isolation as demonstrated by activation and cleavage of caspase-3 yielding the 17 kDa fragment of activated caspase-3 (p17). Data representative of three experiments.
Fig. 3. Matrix reattachment and IEC survival. Isolated IEC crypts were seeded on different components of the extracellular matrix (C I: collagen 1; C VI: collagen VI; FN: fibronectin; LN: laminin). After two and four hours of ex-vivo culture attached (att) and non-attached (sus) IEC were harvested separately and assessed for caspase-3 activation by
Western blot. IEC kept on plastic (co) served as positive control. Attached IEC only contain inactive caspase-3 (p32) while IEC which did not attach and remained in suspension showed evidence of ongoing apoptosis as depicted by caspase-3 activation (p17). Data representative of five experiments.
isolation. These data demonstrate that IEC will remain nonapoptotic as long as they remain attached to the mucosa. IEC isolated at different time points following DTT incubation will initially be non-apoptotic and show no difference in susceptibility to anoikis. Repetition of the isolation procedure can be performed without loss of functionality within IEC.
collagen I plus VI was most effective in ensuring reattachment of primary IEC (data not shown). To assess the role of reattachment for survival floating and attached IEC were harvested separately after 2 and 4 hours of ex-vivo culture. As shown in Figure 3 attached IEC displayed no evidence of caspase-3 activation as only intact non-cleaved caspase-3 can be detected. However, IEC which were unable to reattach showed abundant caspase-3 activation as indicated by detection of the p17 subunit of caspase-3. These data demonstrate the pivotal role of cell-matrix reattachment for survival of primary human IEC. Attempts to increase IEC readhesion by generation of IEC single cells (by a 3-min incubation of the crypts in dispase (1.2 U/ml; Boehringer Mannheim, Mannheim, Germany)) did not increase survival or reattachment, presum-
Reattachment of ex-vivo isolated primary human IEC and survival Since IEC rapidly undergo anoikis following loss of cell anchorage, freshly isolated, non-apoptotic IEC were transferred to matrix-coated tissue culture dishes within 2 ± 3 minutes of detachment. Studies to determine the most suitable matrix for ex-vivo culture had shown that the combination of
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ably due to integrin and cell damage secondary to dispase incubation and prolongation of the time interval for IEC single cell isolation due to dispase incubation and mandatory washing of IEC following dispase incubation (data not shown).
Ex-vivo culture of primary human IEC ± morphology and purity Having demonstrated that IEC which did not reattach will undergo anoikis rather than survive non-adherent IEC were removed by rinsing of the tissue culture vessel after 3 hours of incubation. Subsequently medium was changed every 24 hours and IEC remained attached for 72 hours. Figure 4 displays a photograph of reattached, adherent epithelial cells in ex-vivo culture after 72 hours. Note the typical epithelial character of the flattened well adherent cells forming podocyte-like extrusions and in some instances little monolayers (arrow). However, after 72 hours IEC gradually show evidence of degeneration by shrinking of the cell body, retraction of cell podocytes and eventually detach as non-viable IEC. We have previously shown that cells, freshly isolated by our filter technique are > 95% intestinal epithelial cells (Grossmann et al., 2001). However, a main concern of ex-vivo culture of IEC remains contamination of the culture over time by other cell types ± in particular intestinal fibroblasts. To assess and ensure that the cells maintained in culture remain to be a pure population of IEC we analyzed the cell population in culture once more by flow cytometry after 72 hours. As shown in Figure 5 the cells kept in culture after 72 hours still represent a highly purified population of IEC whereas intestinal fibroblasts and macrophages account for a neglectably small population of cells (< 3%). Fig. 4. Morphology of ex-vivo cultured IEC. IEC were isolated as described above and kept in ex-vivo culture for 72 hours. Cells were studied for morphological features by phase-contrast microscopy (400 magnification). Note the flat spread out epithelial morphology of the cell bodies. Some cells even form small areas with cell aggregates of attached cells (arrow). However, some cells also show vacuolar degeneration at day three of ex-vivo culture, presumably indicating impending terminal differentiation.
Fig. 5. Purity of IEC maintained for 72 hours in ex-vivo culture. IEC were isolated as described above and kept in ex-vivo culture. After 72 hours in culture cells were analyzed by flow cytometry (FACS) to determine the type of adherent cells in culture. More than 90% of cells
Ex-vivo culture of primary human IEC ± viability and functionality Having demonstrated that the cells kept in ex-vivo culture are indeed purified IEC the next goal was to demonstrate viability and functionality of these cells. To assess ex-vivo cultured IEC for evidence of apoptosis cells were harvested at 24, 48 and 72 hours. Neither by DNA electrophoresis (Fig. 6A) nor by flow cytometry (FACS) (Fig. 6B) DNA fragmentation ± the hall-
stained positive for the epithelial cell marker whereas less than 3% of cells stained positive for the macrophage marker or the fibroblast marker AS02. Data representative for five ex-vivo cultures.
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Primary human IEC isolation and culture 267
Fig. 6. Ex-vivo cultivated IEC are non-apoptotic. Freshly isolated IEC were cultivated for 24, 48 and 72 hours as described above. A. DNA was extracted from ex-vivo cultured IEC at indicated time points and analyzed by electrophoresis for evidence of DNA fragmentation. IEC which had been in suspension culture for 180 minutes served as positive control (Co) (M 100-bp DNA marker). Ex-vivo cultivated human IEC show no evidence of DNA fragmentation. Data representative of five experiments. B. At indicated time points IEC were fixed and stained with propidium iodide for DNA content followed by flow cytometry. The data show that ex-vivo cultured IEC are non-apoptotic
as no −sub-G1× apoptotic cell population (arrow) could be detected. C. Ex-vivo cultivated IEC were lysed at indicated time points and activation of caspase-3 was assessed by Western blot. Freshly isolated IEC served as negative control (co 0) and apoptotic IEC (following 180 minutes suspension culture (Co sus)) served as positive control. In exvivo cultivated IEC only the inactive proform of caspase-3 (p32) can be detected, demonstrating that adherent IEC in culture are nonapoptotic. The activation of caspase-3, as depicted by appearance of the p17 subunit can be fully suppressed. Data representative of five experiments.
mark of apoptosis ± could be detected in ex-vivo cultured IEC. In accordance with these findings caspase-3-specific Western blot analysis (Fig. 6C) showed only full-length inactive caspase3 (p32) while the subunit (p17) of caspase-3 indicative of caspase-3 activation could not be detected. These data show that IEC remain non-apoptotic during ex-vivo culture. To demonstrate that IEC are not only non-apoptotic but show actual viability and metabolic activity the MTT assay ± a standard test assessing mitochondrial enzymatic activity ± was performed. As demonstrated in Figure 7 IEC show metabolic activity over the 72 hours of ex-vivo culture. Indeed primary IEC show significantly less metabolic activity (p < 0.05) than the control cells HT-29. This finding may well reflect the
difference between a transformed intestinal epithelial tumor cell and a physiological non-transformed intestinal epithelial cell. These data demonstrate that primary epithelial cells can be kept in a metabolically active state and remain viable during 72 hours of ex-vivo culture. To assess whether primary IEC kept in culture were not only vital but could be used for ex-vivo research further functional studies were performed. In reference to in-vitro findings obtained from studies on the cell line HT-29 (Abreu-Martin et al., 1995) the upregulation of the cell-surface receptor Fas in response to IFN-g incubation was tested in primary IEC. Following an overnight incubation with IFN-g a significant increase in Fas surface expression can be detected in ex-vivo cultivated IEC
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In summary, our data show that primary human IEC purified and cultivated according to the protocol presented are nonapoptotic, viable, metabolically active and suitable for functional studies involving cell metabolism and cytokine stimulation leading to expression of functional cell surface receptors.
Discussion
Fig. 7. Ex-vivo cultivated IEC and metabolic activity. Freshly isolated IEC were cultivated for 24, 48 and 72 hours as described above. IEC were assessed for metabolic activity using the MTT assay. The data show that IEC show persistent metabolic activity at indicated time points. Data show average standard deviation of five experiments. HT-29 tumor cells (HT-29) served as positive control and showed a significantly (* p < 0.05) higher metabolic activity when compared to primary IEC.
(Fig. 8A). To demonstrate that IFN-g incubation leads to upregulation of functional Fas, primary IEC were subsequently incubated with Fas-stimulatory antibody CH-11 to initiate Fas mediated apoptosis (Abreu-Martin et al., 1995). Ex-vivo cultivated IEC readily undergo Fas-mediated apoptosis following incubation with IFN-g and CH-11 as demonstrated by activation of caspase-8, a caspase known to be activated during Fasmediated apoptosis. Interestingly, primary IEC that were not preincubated with IFN-g showed no response to CH-11 (Fig. 8B).
Fig. 8. Ex-vivo cultivated IEC and functionality. IEC were cultivated ex-vivo as described above. A. After 24 hours IEC were incubated with IFN-g, and FAS expression on IEC was analyzed after 12 hours of incubation. Ex-vivo cultivated IEC show functional responsiveness to cytokine stimulation. B. Following IFN-g incubation (20 ng/ml) IEC were treated with control medium or Fas-stimulatory antibody CH-11. After 120 minutes IEC cytosol was obtained and tested for activation of
Apoptosis is a fundamental physiological mechanism inducing programmed death in distinct cells, thereby contributing just as importantly as proliferation to homeostasis of the organism. Colon intestinal epithelial cells proliferate in the stem cell region at the base of the intestinal crypt and subsequently migrate along the basement membrane to the intestinal lumen. As IEC reach the luminal surface, apoptosis is initiated and dying IEC are shed into the lumen as they undergo apoptosis (Potten and Allen, 1977; Gavrieli et al., 1992; Grossmann et al., 2002). The molecular mechanisms underlying the induction of IEC apoptosis in vivo are multifactorial with gradual loss of cell anchorage, a known trigger of apoptosis in various cell types (McGill et al., 1997; Re et al., 1994; Boudreau et al., 1995; Aoshiba et al., 1997) being most likely one factor contributing significantly to this physiological form of cell death at the end of the IEC life cycle (Probstmeier et al., 1990; Beaulieu, 1992; Koretz et al., 1991; Zutter and Santoro, 1990; Riedl et al., 1992). It was not before 1996 that Str‰ter et al. made the key observation that IEC isolation rapidly induces programmed cell death in these cells and furthermore demonstrated that loss of IEC anchorage to extracellular matrix by integrins was responsible for the induction of IEC apoptosis. Further studies could characterize the kinetics and molecular mechanisms
caspase-8 by Western blot. Ex-vivo cultivated IEC are functional as CH-11 incubation leads to activation of caspase-8 (as depicted by appearance of the p43, p41 and p18 subunits) while IEC incubated with medium show no evidence of caspase-8 (p48 and p45 isoforms of procaspase-8) activation. Data representative of three experiments. (Pos positive control, i.e. cytosol from IEC displaying caspase-8 activation)
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underlying IEC anoikis (Frisch and Francis, 1994; Grossmann et al., 1998b, 2001). IEC will initiate the activation of caspases ± key enzymes to initiate and execute apoptosis within a cell ± within minutes of loss of cell anchorage, leading to DNA fragmentation within few hours in essentially all IEC. Indeed, to our knowledge IEC are the most anchorage-dependent cells so far described in the human organism (Grossmann, 2002). Consequently, IEC isolation procedures involving longer incubation periods for enzymatic digestions, several centrifugation steps or even gradient centrifugation are bound to yield IEC undergoing apoptosis. IEC ± once detached ± will rapidly initiate anoikis. Based on our studies on IEC anoikis it became quite clear that the time interval between loss of cell anchorage and reattachment needed to be minimized to overcome induction of anoikis. While our manuscript published in 1998 involved a one-hour incubation in EDTA at 4 8C to inhibit caspase activation and centrifugation steps (Grossmann et al., 1998a) the protocol presented here involves only a ten-minute incubation in EDTA at 37 8C. After these ten minutes the mucosal strips still contain attached, though obviously ™loosened∫ IEC crypts which then are detached from the mucosa by shaking of the container. Following this maneuver the protocol takes advantage of the large crypt structure and quickly harvests all IEC crypts by a filter which can be everted immediately to yield highly purified IEC ready for immediate seeding in a tissue culture well. This rapid and very easy protocol indeed minimizes time of IEC detachment to 2 ± 3 minutes and ensures rapid reattachment of these highly anchorage-dependent cells. Aside from minimizing time for purification the 80-mm filter to harvest the crypts is of great value to ensure purity. Single cells such as fibroblasts, monocytes and lymphocytes will readily pass through the filter. However, the presence of mucus ± especially encountered with tissue samples from the sigmoid and rectum ± in the cell suspension following shaking of the container sometimes would be a problem ™clogging∫ the filter thereby hampering fast filtration and in particular purity. To solve this problem we found that reincubation of the mucosal strips in EDTA and repetition of the critical steps for detachment (see Figure 1) will not only substantially increase the overall yield of isolated IECs but also solves the ™mucus problem∫ as we experienced that only the first IEC harvest ± if at all ± will contain mucus in quantities sufficient to hamper filtration of the IEC. Therefore the repeated incubations in EDTA have substantially improved the IEC yield and solved the ™mucus problem∫ . To enable reattachment tissue culture plates need to be coated with ECM components and our studies confirmed the observation of others (Evans et al., 1994; Whitehead et al., 1987; Buset et al., 1987) that a collagen-based matrix is most efficient in allowing reattachment. As shown impressively by electron microscopy (Str‰ter et al., 1996) only IEC which have contact to the ECM and fully attach will survive while all other IEC will undergo apoptosis, i.e. anoikis. To eliminate dying cells from the culture we chose to wash the plates after three hours of ex-vivo culture as attached IEC by then have formed fully adherent flat cell aggregates while such IEC undergoing anoikis will have dissolved from the crypt and float in the medium. Therefore, after only three hours of ex-vivo culture a fully adherent viable population of purified IEC is available for in vitro studies. A main focus of this manuscript was to demonstrate viability, functionality and maintained purity of IEC during the course of the ex-vivo culture. As a consequence of the high purity of the
Primary human IEC isolation and culture 269
isolate indeed IEC will not be overgrown by ± for instance fibroblasts ± during ex-vivo culture. Furthermore, the population of attached IEC will maintain viability and functionality in terms of responsiveness to cytokine stimulation and upregulation of functional cell-surface receptor expression. Unlike in previous reports, numerous non-subjective tests such as flow cytometry, DNA electrophoresis, Western blot, and MTT assay were employed to confirm this observation. Even though we found that the addition of the PI-3K stimulatory glial-derived neurotrophic factor (data not shown (Steinkamp et al., in press)) will improve survival of IEC during ex-vivo culture it was to date impossible to find conditions prolonging survival of substantial amounts of IEC beyond 3 days of ex-vivo culture. In summary, a further improved and simple method for isolation and short-term ex-vivo culture of viable primary human intestinal epithelial cells displaying important functional properties is presented, making human IEC from surgical specimens amenable for IEC research. Further studies are needed to assess different culture conditions to determine whether senescence of these cells after three days is due to genetic programming reflecting the physiological 3 ± 5-day life cycle of these cells or whether it is due to suboptimal conditions encountered during ex-vivo culture. Acknowledgements. This work is supported by grants ReForM A (J. Grossmann, M. Schultz) and ReForM B (J. Grossmann) grants (Medical Research Funding Program of the University of Regensburg, Regensburg, Germany) as well grants GR1523/3 ± 1 (J. Grossmann) and SFB585 (J. Grossmann, F. Bataille, G. Rogler) from the Deutsche Forschungsgemeinschaft, Bonn, Germany. We wish to thank the Department of Surgery and the Institute of Pathology at the University of Regensburg and the Department of Surgery, St. Josefs Hospital, Regensburg for providing surgical specimens.
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