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The preparation of isolated intestinal crypt cells
Experimental
THE PREPARATION
Cell Research 55 (1969) 257-260
OF ISOLATED
D. D. HARRISON Department of Agricultural
INTESTINAL
CRYPT CELLS
and H. L. WEBSTER
Chemistry, University of Sydney, Sydney, N.S. W., Australia
SUMMARY A rapid and simple method is described for the preparation of isolated crypt cells from the rat intestine. Two successive operations are involved. First, the villous cells are completely removed by a 30 min low-amplitude, high-frequency vibration of the everted, nondilated gut in the presence of 5 mM EDTA-0.14 M NaCl. Second, the crypt cells are specifically dislodged by a maximal dilation of the gut together with further vibrational stress. The product consists of intact crypts in high yield, free of other cellular structures, which can subsequently be disassociated to individual crypt cells by gentle agitation in the presence of 3 mM sodium tetraphenyl borate - 0.15 M NaCl solution.
The process of differentiation of cells from the crypts of Lieberkiihn into mature cells of the mucosal epithelium, coincident with their rapid migration up the villous surface, is a phenomenon of general interest in cellular biology and of particular relevance to studies of intestinal absorption. Experimentation with respect to crypt cells has been limited, up to the present, to examination of in situ material using the techniques of histology and cytochemistry. Recently, Dahlqvist & Nordstriim [l] have reported the activities of some enzymes in material which was obtained by sequential horizontal sectioning of a frozen block of gut. The deepest sections would be expected to contain crypt material associated with elements of the lamina propria. The present communication describes the preparation of intact crypts in high yield from the rat intestine, free of contamination with either villous structures or mature epithelial cells. METHOD
OF PREPARATION
Removal of villous cells The small intestine of a rat (Wistar) was removed, freed of luminal material, and washed with isotonic saline. The gut was cut into four 15 cm lengths and each was tied at one end before everting onto separate rods (diameter 3/16”). The upper margins of the gut lengths were firmly
tied to the rods with surgical silk. For convenience these 4 rods were made part of a single apparatus, being rigidly connected at the opposite ends to a small light metal block fitted with a short attachment rod. The latter allowed the 4 rods to be firmly clamped, vertically, to a Vibromixer Model El (supplied by AG Chemap, Ziirich). A similar technique was described by the authors [2] to prepare brush borders of intestinal cells. However, in the oresent case. the vertical rods were found to be more Satisfactory than a single spiral coil. A cylinder containing about 120 ml of 5 mM EDTA0.14 G NaCl solution was now clamped so as to immerse all gut sections. Vibration of the supporting apparatus was now commenced at an amplitude of 2.0 mm and continued for 30 min. During this period a progressive shedding of small fragments of the mucosa was observed. Phase microscopic examination of this material, collected at intervals, showed small organised pieces of the villous mucosa, whose cells were identified by their characteristic column& shape and by well-developed brush borders in their apical regions. The inclusion of EDTA in the isotonic medium was-found to be significantly effective in the release of these. cellular aggregates from the villous surface.
RESULTS Partial removal of crypts
Measurements of cumulative cell removal from the villi during this period (as judged by protein analysis) showed that after 20 min of vibration the rate of release of material decreased to an insignificant level, which was not affected by the application of increased amplitude of vibration at this stage. Low-power microscopic examination of the Exptl Cell Res 55
258
D. D. Harrison
& H. L. Webster
1. Photomicrograph of the gut surface after vibrational treatment for 30 min. Stained with eosin. x 100. Fig.
mucosal surface of the gut after the vibration period is illustrated in fig. 1. It is seen that the deep clefts at the base of the villi were still occupied by the crypts, which were apparently tightly wedged in position. Further, the villous flaps were denuded of their layer of mucosal cells. It was considered feasible that the crypts could now be released by some procedure which both stretched and dilated the gut, taking advan-
tage of its considerable elasticity. In this way, the crypts would be relieved of lateral retaining pressures. This was achieved by the following procedure, carried out immediately after the vibration period described above, on each of the gut lengths in turn. The suture holding the gut to the rod was carefully cut and the gut, still everted, was gently pushed off the rod. Using fine forceps, the slightly
Fig. 2. Phase-contrast photomicrographs of isolated crypts. x 250. Exptl
Cell
Res 55
Isolation of crypts
i
bulbous end of a 2” length of 3/16” glass tubing was inserted into the open end of the sac, making the attachment firm with a silk suture. The other end of the glass tube was connected to a source of compressed air and the tube was firmly clamped in the vibrator. A 20 g stainless steel mass was now suspended from the lower end of the gut. (It is most important that all handling of the gut, as from the original dissection, be made with particular care to avoid punctures.) The gut was now completely immersed in the EDTA-NaCl solution contained in a cylinder and air was admitted to the sac sufficient to produce maximum inflation. This had the effect of lifting the mass appreciably. At this juncture, no release of cellular material was observed. Vibration of the inflated and stretched gut was now commenced at 2 mm amplitude. An explosive release of material immediately occurred, which was complete in 5 to 10 sec. The suspended particles had a different appearance, seen with the unaided eye, to that of the epithelial material previously collected, being smaller and very uniform in size. The combined yield from the 4 gut lengths was centrifuged for 5 min at 500 g and resuspended as desired in a small volume of the original medium. Fig. 2 illustrates the phase microscopic appear-
259
3. Photomicrograph of thi gut surface after-total treatment for complete crypt removal. Stained with eosin. x 100. Fin.
ante of this material, which contained intact cell complexes whose shape, size and internal organisation established their identity with the crypt structures seen in situ in the clefts of transverse histological gut sections. Careful examination of such preparations indicated the complete absence of mature epithelial cells. Total removal of crypts
The yield of crypt protein, compared to the total protein of the material removed in the first 30 min of vibrational treatment, was 9.1% (ranging from 6.5 to 12.8 “/). This yield, though of biochemically workable size, is an underestimate of the actual proportion of crypts present. The occurrence of minor punctures in the gut, particularly along the mesenteric border, is often hard to avoid. Consequently, inflation pressures tend to be sub-optimal for complete crypt removal. This was supported by the observation that some residual crypts were frequently seen when the completely treated gut was examined by low-power microscopy. If complete crypt removal is desired, the dilation-vibration treatment may be carried out in the following way. After the epithelial cells are removed, an equal length of wet cellulose dialysis tube (Visking, flat width 25/64”), tied off at Exptl
Cell Res 55
260 D. D. Harrison & H. L. Webster this modified treatment is shown in fig. 3. Comparison with fig. 1 indicates that a virtually complete evacuation of the crypt areas has occurred. DISCUSSION
Fig. 4. Phase-contrast photomicrograph cells. x 700.
of isolated crypt
the lower end, is inserted into the still-everted sac. The open ends of both sac and inner tube are tied to the glass tube vibrator attachment as described above. The inner tube, now inflated at approx. 3 lb/sq. inch pressure, supports the sac in the fully-distended state with no interference due to gas leakage. The subsequent vibration is now applied at a setting of 2 mm amplitude, which is retained throughout the gut length, since the support system is sufficiently rigid to preclude damping of the oscillations. Using this modification, the yield of crypts was raised to 16.0 Y0 of the protein removed in the 30 min vibrational treatment (varying from 14.4 to 17.0 %). A low-power photomicrograph of flat strips taken from the everted gut after
Exptl
Cell
Res 55
Isolated crypt cells may be obtained by gentle agitation of crypts suspended in 0.15 M NaCl3 mM TPB (sodium tetraphenyl borate). This potassium-complexing reagent has been shown to be useful in the disassociation of liver tissue, while having a significant effect in the maintenance of the integrity of the cell structure [3]. A photomicrograph of the separated crypt cells is shown in fig. 4. The cells were spheroidal or pear-shaped, noticeably smaller than the mature cells, with a comparatively low cytoplasmicnuclear size ratio. With high power phase microscopic examination, it was observed that some cells possessed very small structures which could correspond to rudimentary brush borders. This rapid and simple preparation of crypt cells allows precise biochemical studies to be made of their chemical components and enzyme activity, which are now being undertaken by the authors. It is considered feasible that such preparations of crypt cells may provide a suitable basis for short-term tissue culture. If so, it may be possible to make a controlled study of the factors which influence the division and differentiation, both morphological and biochemical, of the intestinal cells. This work was supported by a grant from the National Health and Medical Research Council.
REFERENCES 1. Dahlqvist, A & Nordstrom, C, Biochim biophys acta 113 (1966) 624. 2. Harrison, D D & Webster, H L, Biochim biophys acta 93 (1964) 662. 3. Rappaport, C Br Howze, G B, Proc sot exptl biol N Y 121 (1966) 1010. Received October 10, 1968
THE PREPARATION
Cell Research 55 (1969) 257-260
OF ISOLATED
D. D. HARRISON Department of Agricultural
INTESTINAL
CRYPT CELLS
and H. L. WEBSTER
Chemistry, University of Sydney, Sydney, N.S. W., Australia
SUMMARY A rapid and simple method is described for the preparation of isolated crypt cells from the rat intestine. Two successive operations are involved. First, the villous cells are completely removed by a 30 min low-amplitude, high-frequency vibration of the everted, nondilated gut in the presence of 5 mM EDTA-0.14 M NaCl. Second, the crypt cells are specifically dislodged by a maximal dilation of the gut together with further vibrational stress. The product consists of intact crypts in high yield, free of other cellular structures, which can subsequently be disassociated to individual crypt cells by gentle agitation in the presence of 3 mM sodium tetraphenyl borate - 0.15 M NaCl solution.
The process of differentiation of cells from the crypts of Lieberkiihn into mature cells of the mucosal epithelium, coincident with their rapid migration up the villous surface, is a phenomenon of general interest in cellular biology and of particular relevance to studies of intestinal absorption. Experimentation with respect to crypt cells has been limited, up to the present, to examination of in situ material using the techniques of histology and cytochemistry. Recently, Dahlqvist & Nordstriim [l] have reported the activities of some enzymes in material which was obtained by sequential horizontal sectioning of a frozen block of gut. The deepest sections would be expected to contain crypt material associated with elements of the lamina propria. The present communication describes the preparation of intact crypts in high yield from the rat intestine, free of contamination with either villous structures or mature epithelial cells. METHOD
OF PREPARATION
Removal of villous cells The small intestine of a rat (Wistar) was removed, freed of luminal material, and washed with isotonic saline. The gut was cut into four 15 cm lengths and each was tied at one end before everting onto separate rods (diameter 3/16”). The upper margins of the gut lengths were firmly
tied to the rods with surgical silk. For convenience these 4 rods were made part of a single apparatus, being rigidly connected at the opposite ends to a small light metal block fitted with a short attachment rod. The latter allowed the 4 rods to be firmly clamped, vertically, to a Vibromixer Model El (supplied by AG Chemap, Ziirich). A similar technique was described by the authors [2] to prepare brush borders of intestinal cells. However, in the oresent case. the vertical rods were found to be more Satisfactory than a single spiral coil. A cylinder containing about 120 ml of 5 mM EDTA0.14 G NaCl solution was now clamped so as to immerse all gut sections. Vibration of the supporting apparatus was now commenced at an amplitude of 2.0 mm and continued for 30 min. During this period a progressive shedding of small fragments of the mucosa was observed. Phase microscopic examination of this material, collected at intervals, showed small organised pieces of the villous mucosa, whose cells were identified by their characteristic column& shape and by well-developed brush borders in their apical regions. The inclusion of EDTA in the isotonic medium was-found to be significantly effective in the release of these. cellular aggregates from the villous surface.
RESULTS Partial removal of crypts
Measurements of cumulative cell removal from the villi during this period (as judged by protein analysis) showed that after 20 min of vibration the rate of release of material decreased to an insignificant level, which was not affected by the application of increased amplitude of vibration at this stage. Low-power microscopic examination of the Exptl Cell Res 55
258
D. D. Harrison
& H. L. Webster
1. Photomicrograph of the gut surface after vibrational treatment for 30 min. Stained with eosin. x 100. Fig.
mucosal surface of the gut after the vibration period is illustrated in fig. 1. It is seen that the deep clefts at the base of the villi were still occupied by the crypts, which were apparently tightly wedged in position. Further, the villous flaps were denuded of their layer of mucosal cells. It was considered feasible that the crypts could now be released by some procedure which both stretched and dilated the gut, taking advan-
tage of its considerable elasticity. In this way, the crypts would be relieved of lateral retaining pressures. This was achieved by the following procedure, carried out immediately after the vibration period described above, on each of the gut lengths in turn. The suture holding the gut to the rod was carefully cut and the gut, still everted, was gently pushed off the rod. Using fine forceps, the slightly
Fig. 2. Phase-contrast photomicrographs of isolated crypts. x 250. Exptl
Cell
Res 55
Isolation of crypts
i
bulbous end of a 2” length of 3/16” glass tubing was inserted into the open end of the sac, making the attachment firm with a silk suture. The other end of the glass tube was connected to a source of compressed air and the tube was firmly clamped in the vibrator. A 20 g stainless steel mass was now suspended from the lower end of the gut. (It is most important that all handling of the gut, as from the original dissection, be made with particular care to avoid punctures.) The gut was now completely immersed in the EDTA-NaCl solution contained in a cylinder and air was admitted to the sac sufficient to produce maximum inflation. This had the effect of lifting the mass appreciably. At this juncture, no release of cellular material was observed. Vibration of the inflated and stretched gut was now commenced at 2 mm amplitude. An explosive release of material immediately occurred, which was complete in 5 to 10 sec. The suspended particles had a different appearance, seen with the unaided eye, to that of the epithelial material previously collected, being smaller and very uniform in size. The combined yield from the 4 gut lengths was centrifuged for 5 min at 500 g and resuspended as desired in a small volume of the original medium. Fig. 2 illustrates the phase microscopic appear-
259
3. Photomicrograph of thi gut surface after-total treatment for complete crypt removal. Stained with eosin. x 100. Fin.
ante of this material, which contained intact cell complexes whose shape, size and internal organisation established their identity with the crypt structures seen in situ in the clefts of transverse histological gut sections. Careful examination of such preparations indicated the complete absence of mature epithelial cells. Total removal of crypts
The yield of crypt protein, compared to the total protein of the material removed in the first 30 min of vibrational treatment, was 9.1% (ranging from 6.5 to 12.8 “/). This yield, though of biochemically workable size, is an underestimate of the actual proportion of crypts present. The occurrence of minor punctures in the gut, particularly along the mesenteric border, is often hard to avoid. Consequently, inflation pressures tend to be sub-optimal for complete crypt removal. This was supported by the observation that some residual crypts were frequently seen when the completely treated gut was examined by low-power microscopy. If complete crypt removal is desired, the dilation-vibration treatment may be carried out in the following way. After the epithelial cells are removed, an equal length of wet cellulose dialysis tube (Visking, flat width 25/64”), tied off at Exptl
Cell Res 55
260 D. D. Harrison & H. L. Webster this modified treatment is shown in fig. 3. Comparison with fig. 1 indicates that a virtually complete evacuation of the crypt areas has occurred. DISCUSSION
Fig. 4. Phase-contrast photomicrograph cells. x 700.
of isolated crypt
the lower end, is inserted into the still-everted sac. The open ends of both sac and inner tube are tied to the glass tube vibrator attachment as described above. The inner tube, now inflated at approx. 3 lb/sq. inch pressure, supports the sac in the fully-distended state with no interference due to gas leakage. The subsequent vibration is now applied at a setting of 2 mm amplitude, which is retained throughout the gut length, since the support system is sufficiently rigid to preclude damping of the oscillations. Using this modification, the yield of crypts was raised to 16.0 Y0 of the protein removed in the 30 min vibrational treatment (varying from 14.4 to 17.0 %). A low-power photomicrograph of flat strips taken from the everted gut after
Exptl
Cell
Res 55
Isolated crypt cells may be obtained by gentle agitation of crypts suspended in 0.15 M NaCl3 mM TPB (sodium tetraphenyl borate). This potassium-complexing reagent has been shown to be useful in the disassociation of liver tissue, while having a significant effect in the maintenance of the integrity of the cell structure [3]. A photomicrograph of the separated crypt cells is shown in fig. 4. The cells were spheroidal or pear-shaped, noticeably smaller than the mature cells, with a comparatively low cytoplasmicnuclear size ratio. With high power phase microscopic examination, it was observed that some cells possessed very small structures which could correspond to rudimentary brush borders. This rapid and simple preparation of crypt cells allows precise biochemical studies to be made of their chemical components and enzyme activity, which are now being undertaken by the authors. It is considered feasible that such preparations of crypt cells may provide a suitable basis for short-term tissue culture. If so, it may be possible to make a controlled study of the factors which influence the division and differentiation, both morphological and biochemical, of the intestinal cells. This work was supported by a grant from the National Health and Medical Research Council.
REFERENCES 1. Dahlqvist, A & Nordstrom, C, Biochim biophys acta 113 (1966) 624. 2. Harrison, D D & Webster, H L, Biochim biophys acta 93 (1964) 662. 3. Rappaport, C Br Howze, G B, Proc sot exptl biol N Y 121 (1966) 1010. Received October 10, 1968