ISOLATION OF SMALL INTESTINAL CRYPTS.

The cells of the small intestinal mucosa have been isolated to provide a model for the study of enterocyte morphology (Bjorkman et al., 1986), metabolism (Hegazy et al., 1983) and differentiation (Traber et al., 1991) in addition to the investigation of membrane transport processes (Del Castillo & Whittembury, 1987). The small intestinal mucosa of the winter flounder and Necturus maculosa, an aquatic salamander, possess no crypt-like structural invaginations and have been used to prepare apparently homogeneous single cell populations suitable for electrophysiological characterisation (O'Grady et al., 1991; Giraldez et al., 1989). In contrast the heterogeneous transport function of the crypt and villus compartments of the mammalian small intestine is a well established phenomenon (Sundaram et al., 1991; Smith, 1985). Weiser developed a method for the sequential isolation of populations of viable enterocytes from the mammalian small intestine utilising enzymes and chelating agents (Weiser, 1973) which was subsequently improved by the use of hyperosmolar, intracellular-like isolation media (Del Castillo, 1987). Such preparations have been employed to study the electrophysiology of villus-enriched enterocyte populations, identified by their morphology and marker enzyme activity (Sepulveda et al., 1991). However, it is known that enterocytes exhibit a heterogeneity in their resting membrane potentials dependent upon their state of differentiation along the villus axis (Cremaschi et al., 1984). Thus a wide variation in transport activity may be expected even within a relatively enriched single cell population of villus or crypt enterocytes. Moreover although 80% of isolated villus enterocytes have been reported to maintain their structural polarity (Del Castillo, 1987), studies using single cells prepared from the rat and mouse small intestines indicate that the highly polarised distribution of intramembranous particles and enzymes necessary for vectorial transport is lost within minutes of enterocyte dissociation (Ziomek et al., 1980; Bjorkman et al., 1986).

Adult male guinea-pigs (Duncan Hartley strain) were purchased from Tuck and Son, Battlesbridge, Essex, U.K. and were maintained in a temperature-regulated room at 18-21° C and fed ad libitum with a dietary supplementation of vitamin C (Roche). Guinea-pigs with a body weight of between 250-350g were sacrificed by cervical dislocation.

Epithelial fractions isolated as described in 2.2.2. were resuspended in Tris-sucrose buffer to a final volume of 6 ml [buffer composition: 0.25 M sucrose; 4 mM MgCl₂; 0.02 M Trisma base pH 7.4 with 1M HCl] and homogenised at 4° C with an Ultraturrax homogeniser (Jankel & Kunkel, Germany) at 2,500 rpm. The surface layer of lipid was removed from the resultant crude enzyme homogenate using a Pasteur pipette. A 1ml aliquot from each homogenate was spun at 10,000g at 4^oC for 2 min and 0.7 ml of the resulting supernatant was subsequently taken for enzyme assay (Klemperer & Haynes, 1968). The reaction mixture contained 10 m M thymidine (1.96 m Ci/ml ³H thymidine), 5 mM ATP, 2.5 mM MgCl₂ and 0.05 M Tris-HCl, titrated to pH 8.0. Assays were started by the addition of an aliquot of enzyme extract to the above medium (0.1 ml/ml reaction mixture) at 37^oC. The reaction was quenched after 30 min by placing assay tubes in a 100^oC water bath for 2 minutes. The tubes were then put on ice to cool. Denatured protein was removed by centrifugation (2,000g for 10 min). Phosphorylated thymidine was isolated from the "de-proteinised" reaction mixture by binding to Dowex 1x1-100 resin (Gonzalez et al., 1981). Unbound labelled thymidine was removed by repeated washing with 1mM HCl. Bound phosphorylated thymidine was released from the resin with 1M HCl and then counted in a liquid scintillation counter together with appropriate standards. Specific activities were calculated as pmole of [³H] thymidine-P produced per mg protein per minute.

Incubations were performed as originally described by Weiser (1973). Incubations were performed with an assay buffer containing 5 mM MgCl₂ and 50 mM Tris pH 10.1 using 0.1 mM para-nitro-phenyl phosphate (PNPP) as substrate. The reaction was started by the addition of crude homogenate (10-100m l/ml reaction mixture) to pre-equilibrated assay buffer and maintained for 6 minutes at 37° C. Sample assays were performed in duplicate. The reaction was quenched by the addition of 40% w/v trichloroacetic acid, followed by centrifugation at 10,000g for 2 minutes to precipitate any flocculated protein. One ml of supernatant was then mixed with 2.0 ml of 0.4 M NaOH and absorbance readings were taken at 410 nm and related to a 4-nitrophenol standard curve, using PNPP as a blank. Specific activities were then calculated as m mole of PNPP hydrolysed per mg of homogenate protein per minute.

Segments of everted guinea-pig small intestine of 1-2 cm in length were taken at timed intervals and fixed in 4% paraformaldehyde in phosphate buffered saline (PBS, pH 7.2) at 4° C. After 2 hours the segments were washed three times for 20 minutes in PBS before storing the segments in PBS containing 7% sucrose (w/w) and 0.01% azide (pH 7.2) at 4° C. Segments were then dehydrated by immersion in 50% alcohol for 24 hours, 70% alcohol for 10 hours, 90% alcohol for 3 hours and finally by immersion 3 times in absolute alcohol for a total of 5 hours. Segments were then placed in chloroform first for 1 hour and then for 10 hours. The dehydrated segments were then immersed in a fresh paraffin wax bath at 60° C for three periods of 1 hour before being set in a paraffin wax block. Transverse sections of 5m m were cut using a microtome and floated out onto a glass slide.

2.2.7. Staining methods.

The slides were immersed in xylene for 2-3 minutes to remove paraffin wax and then for 2-3 minutes in absolute alcohol to remove the xylene. The slides were subsequently bathed in 70% alcohol for 2-3 minutes, placed under running water and then washed with distilled water prior to staining.

2.2.7.1. Haematoxylin and eosin.

The sections were bathed in Ehrlich's haematoxylin for 10-15 minutes before being rinsed for 5-10 minutes in running water. The sections were subsequently dipped for 3-5 seconds in 70% alcohol containing 1% HCl and then washed again for 5-10 minutes in running water. The slides were dipped in 1% eosin for 5 minutes and then placed under running water until the nuclei appeared blue. The sections were again dehydrated in absolute ethanol and then washed in xylene before being mounted in a xylene-based resin.

2.2.7.2. Periodic acid Schiff and haematoxylin.

Sections were immersed in 1% Periodic acid for 10 minutes, washed under running water for 5 minutes and then rinsed in distilled water. The sections were then bathed in Schiff's reagent for 30 minutes, placed under running water for 5-10 minutes, bathed for 5-10 minutes in haematoxylin before placing under running water again for 5-10 minutes. After 3-5 seconds in 70% alcohol containing 1% HCl the sections were washed under running water for 5-10 minutes before dehydration and mounting as described in 2.2.7.1.

3.3. RESULTS.

3.3.1. Biochemical characterisation of isolated epithelial cell populations.

The relative proportions of crypt and villus enterocytes in the fractions harvested from the everted small intestine were followed by measuring the specific activities of crypt and villus marker enzymes in the isolate. In Fig.3.1. the specific activities of alkaline phosphatase, a marker for mature villus enterocytes, and thymidine kinase, a marker for proliferative crypt enterocytes, are shown for each of the successive fractions collected. Within 4 minutes 85% of the total alkaline phosphatase specific activity isolated was recovered in the medium bathing the everted intestine. During the same period 35% of the total thymidine kinase specific activity was shed. In contrast the fraction collected between minutes 16 and 22 had 0.01% of the total alkaline phosphatase specific activity and 25% of the total thymidine kinase specific activity. Intermediate activities were observed between these times, suggesting that villus and crypt enterocytes had been isolated sequentially.

Fig.2.1.

Histogram showing thymidine kinase and alkaline phosphatase specific activities in epithelial fractions isolated from guinea-pig small intestine against % of total protein isolated in each fraction. Times of successive fractions are given in min.

2.3.2. Effects of isolation protocol upon structure of small intestinal mucosa.

A histological examination of the structure of the intestinal mucosa before, during and after the isolation procedure was performed. Photomicrographs of sections of the everted intestine stained with haematoxylin and eosin are shown in Fig.3.2. prior to entering the isolation buffer (a) and after 10 (b) and 20 (c) minutes of exposure to the isolation buffer. The sections indicate that most of the villus epithelium had been shed from the mucosa within 10 minutes, although crypt epithelium is still present. After 20 minutes the small intestine is almost completely denuded of epithelium.

2.3.3. Morphology and viability of the isolated crypts.

Estimations of the relative proportions of crypt and villus cells in the various fractions were made by light microscopic examination. Phase-contrast light microscopy revealed that early fractions were enriched in caps of villus epithelial cells while later fractions were greatly enriched in morphologically intact, near-cylindrical crypts, with only minor contamination with caps of villus epithelium.

Fig.2.2.

Haematoxylin and eosin stain of sections of guinea-pig small intestine after different periods of exposure to isolation buffer. Transverse sections of guinea-pig small intestine showing extent of epithelium in intestinal segments fixed in 4% glutaraldehyde immediately before (a), and after 10 (b) and 20 (c) minutes of exposure to isolation buffer.

A phase-contrast photomicrograph of an isolated small intestinal crypt is shown in Fig.2.3., in which the smooth basolateral membrane and narrow lumen can be discerned. Measurements taken from 25 isolated crypts gave a mean "depth" and width of 211 ± 6 m m and 76 ± 3 m m respectively (means ± SEM). A mean depth of 179 m m has been reported previously from measurements on glutaraldehyde-fixed guinea-pig small intestine (Smith et al., 1984). The virtual lumen is enclosed at one end by a sheath of crypt cells, often referred to as the ground region (G), whilst the other end opens out at the surface region (S) into the external environment. Another characteristic feature is the progressive decrease in crypt diameter from the ground region, through the mid-region (M) to the surface region. These morphological criteria are later used as a positional reference for the enterocyte within the crypt during electrophysiological measurements.

Trypan blue was initially completely excluded from 80% of all morphologically intact crypts that could normally be identified as "healthy" by their birefrigence under phase-contrast microscopy. Stained crypt cells were granular and lacked a smooth basolateral membrane and birefringency. Crypts remained viable for up to 60 min at 22-25° C and for 6 to 8 hours when maintained on ice. The high viability of crypts isolated by this method is also suggested by measurements of crypt membrane potential and the reversible responses to secretagogues which are described later.

Fig.2.3.

Morphology of a small intestinal crypt isolated from guinea pig jejunum. Phase-contrast photomicrograph of an isolated small intestinal crypt with the central lumen (L), ground (G), middle (M) and surface (S) regions indicated. B. Periodic acid Schiff and haematoxylin stain of transverse section of small intestinal crypt. The nuclei located towards the basal pole of the cell are stained blue, whilst the abundant mucin-containing goblet cells (GC) which can be seen releasing their contents into the lumen are stained scarlet. The ground region (G) and the surrounding lamina propria (LP) are labelled. Scale bars represent 25m m.

The low temperature isolation method avoids the loss of epithelial polarity due to compromised cell adhesion and membrane integrity that is associated with isolation methods performed at 37° C (Flint et al., 1991). More importantly for functional studies the internalisation of surface receptors in enterocytes that occurs at 37° C is prevented by performing the isolation at low temperature (Gallo-Payet & Hugon, 1985). Additionally, the loss of relatively unstable mRNA transcripts or their protein products that occurs at 37° C does not occur at 4° C. The preparation described here has been used to show the differential patterns of expression of angiotensin II receptor mRNA and protein between villus and crypt units isolated from rat jejunum (T.Vaughan, personal communication). Similarly the relative enrichment of Na⁺-glucose carrier mRNA in villus tip fractions, with a total absence of message in the isolated crypt fraction, has been demonstrated by Northern blot analysis of mRNA isolated from guinea-pig jejunal epithelial fractions (T.Vaughan, personal communication).

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