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WO2000003002A2 - Preparations de cellules epitheliales humaines pures et viables a partir de tissu digestif - Google Patents

Preparations de cellules epitheliales humaines pures et viables a partir de tissu digestif Download PDF

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WO2000003002A2
WO2000003002A2 PCT/CA1999/000621 CA9900621W WO0003002A2 WO 2000003002 A2 WO2000003002 A2 WO 2000003002A2 CA 9900621 W CA9900621 W CA 9900621W WO 0003002 A2 WO0003002 A2 WO 0003002A2
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cells
human
culture
cell
epithelial
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WO2000003002A3 (fr
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Jean-François BEAULIEU
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Universite de Sherbrooke
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0679—Cells of the gastro-intestinal tract
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the present invention relates to an in vitro model system of human digestive epithelial cells which is pure, viable, functional and representative of normal human digestive epithelium and can be used to test molecules in order to assess their effects on physiology, growth and homeostasy.
  • the present invention further relates to a simple, non-enzymatic method to prepare such pure, viable and functional human epithelial cells from digestive tissue.
  • the epithelium of the small intestine is a highly dynamic system particularly well suited for analyzing key biological phenomena such as cell proliferation, migration, differentiation and apoptosis. Indeed, within its functional unit, the crypt-villus axis, the epithelium is spatially separated into proliferative and differentiating intestinal cells, located respectively in the lower and upper crypt regions, and functional cells lying on the villus [1 ,2,8,20].
  • the renewal of the intestinal epithelium is a relatively complex process which is likely to be regulated by a number of factors including hormones, growth factors, cytokines, cell-cell and cell-matrix interactions [3-5, 7, 37].
  • the gastric epithelium assumes important key functions such as a protection barrier against acid retrodiffusion or bacterial invasion, an essential role in epithelial restitution following injuries and the digestion of dietary proteins and t ⁇ glycerides
  • the gastric functional unit, the foveolus-gland axis is composed of four distinct compartments namely pit isthmus, neck and base of the gland
  • the stem cell population responsible for the renewal of the entire gastric epithelium is located in the isthmus After division, the cells migrating upward will differentiate and renew pit and surface mucous epithelial cells while the ones migrating downward will renew chief, parietal, endocrine and caveolated cells [52-56]
  • This highly dynamic system would be particularly well suited for analyzing key biological phenomena such as cell proliferation, migration and differentiation
  • the present invention relates to a human epithelial cell model system derived from digestive tissue which is representative of the normal digestive tissue situation
  • the present invention also relates to a method to purify and isolate epithelial cells from human mesenchymal digestive tissue
  • the present invention relates to a simple procedure that allows an efficient dissociation of human digestive tissue into pure epithelial and corresponding mesenchymal fractions
  • the invention relates to the dissociation of human intestine into a pure epithelial and corresponding mesenchymal fractions
  • the procedure was found particularly well suited to the fetal intestine at mid-gestation, a period at which the mucosa morphologically and functionally resembles that of the adult [20]
  • Analysis of the fractions for the presence of transcripts for a number of extracellular matrix molecules revealed that the epithelium produces most of the formal BM molecules while BM-associated molecules are mostly of mesenchymal origin
  • the method of purification and isolation of epithelial cell from digestive tissue is used on gastric tissue and shows that gastric epithelium can be easily and efficiently isolated from the developing human gastric mucosa
  • the present invention also relates to an in vitro cell model system of a normal crypt-villus axis of human intestine comprising a primary cell culture of human intestinal epithelium which is functional and viable for more than two days in culture a crypt-like cell line comparable to undifferentiated proliferate cells of the lower crypt, and a crypt-hke cell line which can be induced to differentiate, comparable to cells of the upper half of the crypt
  • the cryp-hke cell lines can be chosen by a person of ordinary skill to which the present invention pertains amongst the known crypt-hke cell lines Non-limiting examples thereof include HIEC, tsHF1 and H-4 [98]
  • the procedure of the present invention to purify and isolate epithelium from digestive tissue is also demonstrated herein with gastric mucosa It is indeed demonstrated that gast ⁇ c epithelium can be easily and efficiently isolated from the developing human gastric mucosa with MatnsperseTM
  • the resulting preparations consisting of intact epithelial cell sheets or large aggregates can be maintained as p ⁇ mary cultures in minimal culture conditions for up to 2 weeks
  • the above non-enzymatic dissociating procedure appeared to be very efficient as for the practicability of isolation as well as preserving cellular integrity and viability
  • MatnsperseTM, a non-enzymatic dissociation solution allowed the isolation of substantially all epithelial cell types found in gastric epithelium and their maintenance in primary culture for up to two weeks
  • the cell preparation was shown to be substantially pure viable, functional and representative of intact gastric epithelium
  • the primary cultures of gastric epithelial cells of the present invention can thus be used to analysis of key biological phenomena such as cell migration and differention, cell
  • the method of digestive tissue epithelium preparation of the present invention is a non-enzymatic method which yields a cell preparation which can grow for up to 10 days
  • the method of purification and isolation of epithelium from human digestive tissue comprises an incubation of human digestive tissue with MatnsperseTM
  • the non-enzymatic method of separation of epithelium from mesenchymal cells significantly minimizes the contamination of the resulting epithelial cell preparation with molecules and/or cells such that they are not representative of intact or normal gastric epithelium
  • a method to separate a human digestive tissue epithelium from its underlying mesenchyme comprising the step of incubating the human digestive tissue with an effective amount of MatnsperseTM for a sufficient time to enable
  • an in vitro cell model system of a normal crypt-villus axis of human intestine comprising a p ⁇ mary cell culture, a crypt-like cell line comparable to undifferentiated proliferate cells of the lower crypt, and a crypt-hke cell line which can be induced to differentiate, comparable to cells of the upper half of the crypt
  • a method of generating functional chief cells from human fetal stomach comprising an incubation of the human fetal stomach with an effective amount of MatnsperseTM and for a sufficient time to enable an obtention of the functional chief cells in a primary cell culture of gastric epithelium.
  • a method of preparing primary epithelial cells from human digestive tissue comprising an incubation of the human digestive tissue with an effective amount of MatnsperseTM and for a sufficient time to enable the preparation of primary cells of human digestive epithelium which are substantially free of mesenchyme, and can be maintained in culture for more than 2 days
  • MatnsperseTM an effective amount of MatnsperseTM and for a sufficient time to enable the preparation of primary cells of human digestive epithelium which are substantially free of mesenchyme, and can be maintained in culture for more than 2 days
  • intact cells refer to the fact that the primary cultures of human digestive epithelium, in accordance with the present invention, substantially express the same markers, as assessed biochemically or morphologically, as their epithelial cells counterparts found in the intact organ
  • time of incubation of the digestive tissue in Matnsperse can be adapted to meet particular needs (i e total vs partial separation of the epithelium from the mesenchymal tissue)
  • time of incubation required for complete dissociation varies between gastric and intestine tissues
  • the present invention provides numerous methods to assess the level of separation of the epithelium and mesenchymal tissues
  • the person of ordinary skill will be able to adapt the incubation time and concentration of Matnsperse to meet particular needs
  • the primary cultures of the present invention are viable in culture for a substantially higher time than that of the prior art (about 2 days) Indeed, the primary cultures of the present invention remain viable and functional in culture for at least 3 days, preferably 5 to 10 days and routinely for about 12 days
  • the primary cells of the present invention will have been "transfected" by exogenous or heterologous DNA (e g a DNA construct) when such DNA has been introduced inside the cell
  • the transfecting DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell
  • a stably transfected cell is one in which the transfecting DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transfecting DNA Transfection methods are well known in the art (Sambrook et al , 1989, supra, Ausubel et al , 1994 supra)
  • the primary cell cultures of the present invention could be transfected as well known to the person of ordinary skill in order to adapt them to particular needs
  • Figure 1 shows the representative phase contrast micrographs of the iuminal aspect of a human fetal small intestine following a treatment of 9 hours with MatnsperseTM Before agitation the intestinal epithelium remains in place (A) After a gentle agitation, note the complete dissociation of the entire epithelial lining (B) from the remaining underlying mesenchyme (C) (Original magnification, x19)
  • FIG. 2 shows the western blot analysis of intact small intestine and Mat ⁇ sperse-dissociated intestinal epithelial and mesenchymal fractions with tissue-specific markers
  • Samples of intact intestine (lane 1) and corresponding mesenchymal (lane 2) and epithelial fractions were separated by SDS-PAGE and transferred to nitrocellulose membrane for immunodetection Sucrase-isomaltase (SI), E-cadhe ⁇ n (E-cad), keratin 18 (K18) and the MIM-1/39 antigen were exclusively detected in the intact intestine and epithelial fraction while vimentin (VIM) and ⁇ -smooth muscle actin ( ⁇ SMA) were present only in the intact and the mesenchymal fraction
  • SI immunodetection Sucrase-isomaltase
  • E-cad E-cadhe ⁇ n
  • K18 keratin 18
  • MIM-1/39 antigen were exclusively detected in the intact intestine and
  • Figure 3 shows a representative Northern blot analysis for the detection of transcripts encoding extracellular matrix molecules (A) and markers (B) in the intact fetal small intestine at mid-gestation (lane 1) and in corresponding isolated mesenchyme (lane 2) and epithelial fractions (lane 3)
  • Transcripts analyzed were the ⁇ 1 (IV) collagen chain (C ⁇ 1 (IV)), laminin ⁇ 1 chain (Ln ⁇ 1), fibronectin (Fn), deco ⁇ n (Dc) SPARC/osteonectin (SPARC), E-cadhe ⁇ n (E-cad), sucrase-isomaltase (SI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and
  • Figure 4 shows a representative RT-PCR analysis of transcripts encoding extracellular matrix molecules (A) and markers (B) in the intact fetal small intestine at mid-gestation (lane 1) and in the corresponding isolated mesenchyme (lane 2) and epithelial fractions (lane 3) cDNAs were transcribed from total RNAs and amplified with primer sets specific for laminin ⁇ 1 (Ln ⁇ 1) and ⁇ 2 (Ln ⁇ 2) chains collagen ⁇ 5(IV) (C ⁇ 5(IV)) and ⁇ 6(IV) (C 6(IV)) chains, tenascin-C (Tn-C), E-cadhe ⁇ n (E-cad), sucrase-isomaltase (SI) and S14
  • Figure 6 shows the phase-contrast morphology of p ⁇ mary cultures of gastric epithelial cells A) After 1 5 day of culture, epithelial colonies are intensely spreading and fusing (x10) B) By day 3, a confluent monolayer of polyhedral and irregularly-shaped cells is obtained (x10) C) After 7 day, the monolayer remains confluent (x10) D) After 14 days, the monolayer remains intact but vacuohzation is visible in some cell clusters (filled arrow) (x10)
  • Figure 7 shows the growth characteristics of gastric epithelial cells
  • A Cell numbers were determined at different culture intervals by dissociation (trypsin EDTA) of cultured cells and hematocytometer counting The number of cells rapidly increases (two fold) during the first 48 hours
  • B 3 H- thymidine incorporation into total DNA
  • An increase in incorporation of radiolabeled precursor is noted after 1 5 day and peaks after 2 days of culture Values represent the mean ⁇ SEM of 3 separate and independent cultures
  • Figure 8 shows the immunodetection of tissue-specific cell markers in gastric epithelial cells After 1 5 day of plating, representative indirect immunofluorescence micrographs of gastric cells stain for the detection of kerat ⁇ n-18 (A), vimentin (B), ZO-1 (C) and E-cadhe ⁇ n (D) Gastric epithelial cells are found positive for kerat ⁇ n-18 while vimentin was detected only in some cells at the periphery of colonies Tight-junction and zonula adhere
  • FIG. 9 shows the immunodetection of growth factor receptors (1 5 day of culture)
  • D) KGF-R or K-sam protein These receptors known to be expressed in fetal gastric epithelial cells in vivo are readily detected in cultured cells and exhibit their characteristic cellular patterns membrane-associated EGF-R, less abundant IGF1-R, membrane-bound and cytoplasmic for c-met and K-sam (original magnifications A-D, x40)
  • Figure 10 shows the histochemical analysis of functional markers
  • FIG. 11 shows the immunodetection of human pepsinogen (Pg5) in gastric ceils 1 5 day after plating
  • a subpopulation of cultured cells shows an intense granular staining (filled arrows) (x40)
  • Densitomet ⁇ c analysis illustrates the relative levels of Pg5 protein expressed in arbitrary units (AU) as the values were normalized to kerat ⁇ n-18 signals Values represent the mean ⁇ SEM of 4 separate and independent experiments
  • Figure 12 shows the immunodetection of human gastric lipase (HGL) un gastric cells 1 5 day after plating A) Similarly as in Fig 11 , a subpopulation of cells is immunoreactive for anti-HGL staining (x40) Insert shows a positive chief cell at higher magnification (x40) B) Representative Western blot analysis of cultured cells at 1 5, 3 and 5 days
  • Figure 13 shows the pepsinogen (Pg5) and hpase (HGL) activity in gastric epithelial cells Cumulative activity of Pg5 (A) and HGL (B) determined in cells and media after 1 5, 3, and 5 days of culture Synthesis and secretion profiles of Pg5 (C) and HGL (D) activities measured in cells (g) and in medium (c) between culture intervals (1 5-3 days, 3-5 days)
  • Pg5 pepsinogen
  • HGL hpase
  • Figure 14 shows the effect of EGF on Pg5 and HGL activity and on HGL-mRNA expression
  • E Densitomet ⁇ c analysis expressed in arbitrary units (AU) is normalized to GAPDH signals used as control Values represent the mean ⁇ SEM of 3 separate and independent experiments
  • MatnsperseTM a commercial non- enzymatic preparation initially designed to isolate epithelial cells grown on EHS biomat ⁇ x, appeared to be instrumental in both the efficiency of the isolation procedure and cell viability preservation
  • procedures designed to isolate intestinal epithelial cells have included various dissociating enzymes such as pronase, dispase, collagenase, etc alone or in combination that invariably, with the exception of thermolysin when used under strict conditions [44], resulted in some degree of mesenchymal cell contamination and limited epithelial cell recoveries [19 44, 98]
  • MatnsperseTM under the conditions described herein, allowed the isolation of a large proportion, if not all, of the epithelium free of mesenchymal cells as assessed by phase contrast microscopy and
  • PCDE represents a normal counterpart for the two widely used colon adenocarcinoma cell models HT-29 and Caco-2 under their differentiated state [12,111]
  • PCDE will allow the in vitro reproduction of the entire normal crypt-villus axis, each of these models representing one of the three main cell populations the HIEC, comparable to the undifferentiated proliferative cells of the lower crypt [111], the tsHFI cells, which can be induced to undertake differentiation through a process comparable to the one occurring in the upper half of the crypt in vivo [97], and the fully functional villus-hke PCDE
  • PCDE can be used advantageously for a number of purposes including hormone and growth factor regulatory influences as well as cell-cell and cell- matrix interactions
  • hormone and growth factor regulatory influences as well as cell-cell and cell- matrix interactions
  • PCDE appears as an interesting normal alternative to the currently used models IEC-6, Caco-2, T-84 or HT-29 [112,113] allowing the identification of relevant factors that stimulate epithelial cell migration
  • Other potential applications for PCDE are numerous and could include the analysis of drug and nutrient transport and metabolism [114-115] and the study of microorganism- intestinal epithelial cell interactions [116]
  • the present invention teaches the first attempt to determine the tissular origin of extracellular matrix molecules from the intact human intestine
  • the knowledge of the origin of epithelial basement membrane components in the intestine comes mainly from experiments performed on laboratory animals [17,33-36]
  • results from experimental animals cannot always be directly extrapolated to the human
  • fundamental differences in the regulation of gene expression during intestinal development and along the crypt-villus axis in the adult have been found between man and animal models [20,37,38]
  • Differences in the distribution of basement membrane components have been also observed [6-8]
  • human laminin- 1 has been found to be predominantly associated with the differentiated enterocytes lining the villus [39] while it has been found to be restricted to the proliferative compartment in rodents [40
  • Co- culture of human intestinal epithelial cells with fibroblasts has also been used to analyze epithelial-mesenchymal interactions in vitro [41-43]
  • the epithelium of the gastric foveolus-gland axis is a highly dynamic structure particularly well suited for analyzing key biological phenomena such as cell migration and differentiation, cell-matrix interactions, and apoptosis
  • MatnsperseTM a non-enzymatic dissociating solution
  • Indirect immunofluorescence and Western blot analyses confirmed the purity of epithelial cells
  • the primary cultures were composed of mucus-secreting cells, zymogenic chief cells and parietal cells Chief cells were able to produce and secrete and respond to certain growth factors Taken altogether, these data show for the first time that primary cultures of gastric epithelial cells containing viable and functional chief cells can be generated from the human fetal stomach
  • EXAMPLE 1 Sampling of intestinal tissue, epithelial-mesenchymal dissociation thereof and RNA extraction. Specimen of small intestine and colon from 22 fetuses ranging from 15 to 20 weeks of age (post-fertilization) were obtained after legal abortion The project was in accordance with a protocol approved by the Institutional Human Research Review Committee for the use of human material Only specimen obtained rapidly were used Pure pools of epithelial cells and mesenchyme were obtained from small intestine (jejunum and ileum) and colon according to the following procedure The serosa and muscula ⁇ slitis were first removed and the remaining segments were opened longitudinally, washed in phosphate buffered saline (PBS) and cut into 5 mm fragments Small intestinal and colonic fragments were transferred to ice-cold MatnsperseTM solution (Collaborative Biomedical Products, Becton Dickenson Labware, Mississauga, Ontario, Canada) and incubated at 4°C for about 8-1 Oh without
  • RNA samples were subjected to agarose gel electrophoresis with formaldehyde and transferred for Northern blot analysis to nylon membranes (Nytran, Schleicher and Schuell) Equal RNA loading (15 ⁇ g) was confirmed by hybridization to a rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene control Prehyb ⁇ dization and hybridization were performed as described previously [18]
  • Hybridizations were performed with the followed random primed 3 P-labeled cDNA probes (Multip ⁇ meTM Kit, Amersham, Oakville, Ontario) 1 3 kb Pst 1 rat GAPDH fragment [23], 1 7 kb Pst I human type IV collagen cDNA fragment [24], 1 7 kb Bgl II human sucrase-isomaltase cDNA fragment [25], 4 6 kb EcoR I murine laminin B1 chain cDNA fragment [26], 4 5 kb Hind III human fibronectin cDNA fragment [27], 1 kb Apa I and Pst I human deco ⁇ n fragment (Gibco BRL) and a 2 2 kb BamH I pig SPARC fragment (kindly provided by Dr J Sodek)
  • the E-cadhe ⁇ n probe was prepared from polymerase chain reaction (PCR) amplification of cDNA [28] prepared from total RNA extracted from a 18-week-old human fetal intestine
  • the sense primer 5'-ctggtctgagtcttcgttcc-3' and the antisense primer 5'-tcctgacagccgagaaaggc-3' were used
  • the sense primer hybridizes with the Fnlll 5 (bases 3290 to 3309) domain, and the antisense primer hyb ⁇ dyze with Fnlll 6 (bases 5410 to 5429) (Genebank #A55618)
  • These primers were expected to generate a major 230 bp product
  • Single stranded cDNA was amplified in PCR buffer containing 1 ⁇ M of both primers for 28 cycles of denaturation (1 m ⁇ n at 93°C) and annealing/extension (1 mm at 61 °C and 3 mm at 72°C) in a thermal cycler (Perkin-Elmer DNA thermal cycler model 480) in the presence of 250 ⁇ M dNTP's and 2 5 U of Taq
  • Tissues from 48 fetuses varying in age from 17 to 20 weeks of gestation were obtained from normal elective pregnancy terminations No tissue was collected from cases associated with known fetal abnormality or fetal death Studies were approved by the Institutional Human Subject Review Board
  • the stomach was brought to the culture room, immersed in dissection medium i e Leibovitz L-15 plus gentamycm and nystatm (40 g/mL each) and prepared within 30 minutes at room temperature
  • Cardiac and pylo ⁇ c segments were removed from the stomach leaving the body and fundic regions Tissue specimens were cut into explants (3x3mm 2 ) and rinsed with dissection medium [74]
  • the gastric epithelium was dissociated using a new non-enzymatic technique based on a procedure to recover cells grown on Mat ⁇ gelTM as previously applied to human fetal small intestine (see above and [75]) Explants were immersed in ice
  • Protein markers associated with human fetal gastric epithelial cells were analyzed by indirect immunofluorescence as described above [58,75] A number of antibodies were used antibodies directed to the 1) human growth factor receptors EGF/TGF ⁇ -R (1 100, Upstate Biotechnology, Lake Placid, NY), IGF1-R (1 25, Oncogene Research distributed by Cedarlane, Hornby, Ontario), HGF-R or c-met (1 200, Santa Cruz Biotechnology, Santa Cruz, CA) and KGF-R or K-sam (1 100, kindly given by Dr M Terada, National Cancer Center Research Institute, Tokyo, Japan), 2) digestive enzymes of gastric chief cells human fundic pepsinogen (Pg5) and human gastric pase (HGL) (1 150 and 1 4000 respectively two polyclonal antisera obtained from R Verger, A DeCaro and F Car ⁇ ere, INSERM Marseille, France), 3) the anti- gast ⁇ c H7K + ATPase of parietal cells (1 800, Calbiochem, LaJolla, CA
  • RNA used in Northern blot analysis was isolated as described [81] Equivalent amounts of RNA (20 ⁇ g) were used for electrophoresis through 1 % agarose gels containing 6% formaldehyde and blotted onto nylon membranes (Hybond-N, Amersham) Equal RNA loading was confirmed by ethidium bromide staining and by hybridization to a rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe [30] Membranes hybridized with DNA probes were processed, blotted dry and autoradiographed For densitomet ⁇ c analysis, signals were normalized to those of the GAPDH control
  • DNA synthesis measurements were performed as described elsewhere [107] Briefly, isolated epithelial fractions, Caco-2/15 and HIEC-6 cells were plated onto 60 mm dishes After 48 h, 2 ⁇ Ci/ml of [ 3 H]thym ⁇ d ⁇ ne was added and incubated for a further 12 h Cells were then washed twice and DNA was precipitated with two subsequent treatments of 10% t ⁇ chloroacetic acid After solubihzation with 0 3 M NaOH and neutralization with 1 5 N HCl, total [ 3 H]thym ⁇ d ⁇ ne incorporation was evaluated using a Beckman LS 6800 scintillation counter
  • Dissociation of the intestinal epithelium from the mesenchyme was achieved by using MatrisperseTM a dissociating solution initially designed to recover epithelial cells grown on EHS matrix As shown in Fig 1 , small intestinal fragments remain relatively unaltered after 8-11 h of incubation at 4°C without agitation in MatrisperseTM other than a swollen appearance of the vilh (Fig 1A) However, gentle agitation of the medium results in a rapid dissociation of the entire epithelial lining (Fig 1 B) from the underlying mesenchyme (Fig 1 C) Comparable epithelial-mesenchymal dissociation was obtained on 16-20 week-old jejunum and ileum The only difference noted was in the time required for dissociation (jejunum 10-11h, ileum 8-9 h)
  • Intestinal epithelial cell cultures were further characterized by electron microscopy and indirect immunofluorescence 6-7 days after the plating These studies showed that the cultures are composed of both goblet and absorptive cells (not shown) exhibiting ultrastructural characteristics similar to those found in the intact villus epithelium, including well organized brush borders and terminal webs at the lummal aspect of absorptive cells as well as typical junctional complexes
  • the immunodetection of various markers confirmed the relative purity of the epithelial cultures and their integrity (not shown) All cells were found positive for keratin 18 with the exception of goblet cells, which were identified with an anti-mucm antibody while mostly negative for the mesenchymal cell marker vimentin
  • Expression of various junctional molecules found normally in epithelial cells was also analyzed Expression of the ZO-1 protein, desmosomal protein and E-cadhe ⁇ n were all localized homogeneously at the cell-cell interfaces
  • the expression of a number of intestinal cell markers was investigated Su
  • a crypt-cell marker the MIM-1/39 antigen
  • the proliferative potential of these cultures was then investigated for DNA synthesis 48 h after the plating, a stage when colonies are rapidly expanding.
  • [ 3 H] thymidine incorporation in these cultures was found negligible as compared to that found in proliferating epithelial intestinal cells such as HIEC-6 cells and subconfluent Caco-2 cells.
  • EXAMPLE 18 Pure human intestinal epithelium cell cultures as a model system for normal human intestine
  • BM components such as the laminins and type IV collagens but also BM-associated molecules such as fibronectin, tenascin-C and decorin [7].
  • fibronectin was found to be of dual origin Fibronectin is expressed at relatively high levels in the developing human small intestine, being distributed throughout the mesenchyme including the epithelial-mesenchymal interface (45, see Table 2)
  • intestinal epithelial cells can synthesize fibronectin was first suggested by its identification in the basal lamina of the intact tissue [46] Its expression by undifferentiated intestinal epithelial cells in vitro [47,48] supported this possibility
  • the fibronectin transcript has been shown to be present in epithelial fractions However, its relatively low amounts as compared to other molecules exhibiting a dual origin, such as the laminin
  • EXAMPLE 19 Dissociation kinetics of gastric epithelium from explants of human fetal stomach
  • the Mat ⁇ sperseTM-generated epithelial cell cultures consisted of mucous-type (about 60%) and parietal cells (about 5%) as well as a significant population of chief cells (about 30%) as revealed by Bowie staining and immunological analyses
  • the human chief cells are characterized by the presence of both pepsinogen and gastric lipase associated with secretory granules as previously reported in human gastric glands [87] This is particularly important since the understanding of the regulatory mechanisms of human chief cell functions has remained fragmentary because available animal models either lack gastric lipase (rat/mouse [88]) or have both gastric digestive enzymes located in different cell types (dog [89], cat [90], rabbit [91], guinea pig [92])
  • human chief cells retained their capacity to express and secrete Pg5 and HGL in vitro Interestingly, differential expression and secretion profiles for both digestive enzymes in
  • gastric epithelial primary cultures representative of the intact foveolus-gland axis can be generated from human fetal stomach and maintained viable, even in absence of added biological matrix
  • the human fetal gastric gland is representative of the adult gastric gland since the functional compartments are already fully determined, all differentiated epithelial cell types are in place, all functional markers as well as known hormone and growth factor receptors are expressed and the proper distribution along the foveolus-gland axis of extracellular matrix components and integ ⁇ n-type receptors is established [58,83] Therefore, eventhough the fetal glands are smaller than the adult ones they already exhibit the adult functional characteristics as soon as 15 weeks of gestation
  • This culture system can be used to unravel the cellular and molecular events underlying specific hormone and growth factor regulatory influences, cell-cell and cell-matrix interactions involved in the maintenance of the normal physiology of the foveolus-gland axis or implicated in pathological conditions Another important
  • the primary cultures of fetal gastric epithelium of the present invention may provide a number of advantages such as: (1) initial attachment and survival does not require the presence of a biological substratum whereas conventional systems use soluble fibronectin or collagen, (2) for the first time, they contain a significant fraction of functional chief cells able to synthesize and secrete digestive enzymes, namely pepsin and gastric lipase, (3) cell types are heterogenous and are thus the only model representative of the intact gastric epithelium, as opposed to cultures generated after enzymatic dissociation which are composed of surface mucous cells mostly, (4) proliferative cells are present; fetal bovine serum (FBS) well supports their growth at normal concentration (10% v:w), and (5) confluency is reached after 3-4 days that allows the formation of a compact monolayer sealed with tight junctions: an epithelial barrier.
  • FBS fetal bovine serum
  • Insulinlike growth factor-binding protein modulates the growth response to insulinlike growth factor 1 by human gastric cancer cells Gastroenterology 104, 1595-1604 Sanders, M J , Amman, D A Ayalon, A , and Soil, A H (1983) Regulation of pepsinogen release from canine chief cells in primary monolayer culture Am J Physiol 245, G641-G646 Rattner, D W , Ito, S , Rutten M J , and Silen, W (1985) A rapid method for culturing guinea pig gastric mucous cell monolayers In Vitro Cell Dev Biol 21, 453-462 Chew, C (1994) Parietal cell culture new

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une méthode de séparation d'un épithélium de tissu digestif humain de son mésenchyme sous-jacent comprenant l'étape consistant à incuber le tissu digestif humain avec une solution à 100 % de MatrisperseTM pendant 8 à 20 heures afin de permettre la séparation de l'épithélium du mésenchyme. Dans les modes de réalisation particulièrement préférés de l'invention, le tissu digestif est du tissu intestinal ou gastrique. L'invention concerne également des cultures de cellules épithéliales primaires dérivées de tissu digestif humain ayant été obtenues par un traitement non enzymatique de tissu digestif et lesquelles sont pures, fonctionnelles et viables en culture pendant au moins deux jours en l'absence d'un support mésenchymateux . Dans un mode de réalisation particulier, la culture de cellules épithéliales intestinales comprend des cellules caliciformes et des cellules absorbantes ayant conservé toutes les caractéristiques rencontrées in vivo dans l'épithélium intestinal dont on a dérivé la culture cellulaire primaire. Dans un autre mode de réalisation, la culture de cellules épithéliales gastriques comprend des cellules sécrétant du mucus, des cellules pariétales et une quantité significative de cellules principales zymogènes. De plus, l'invention concerne un système de modèle cellulaire in vitro d'un axe cryptes-villosités normal de l'intestin humain comprenant une culture cellulaire primaire d'épithélium intestinal humain laquelle est fonctionnelle et viable pendant plus de deux jours en culture, une lignée cellulaire de type crypte comparable à des cellules prolifératives non différenciées de la crypte inférieure, et une lignée cellulaire de type crypte pouvant être induite pour différencier, comparable à des cellules de la moitié supérieure de la crypte. L'invention concerne l'utilisation des cultures cellulaires primaires de la présente invention en tant que systèmes de modèles pour identifier des médicaments et des composés les affectant.
PCT/CA1999/000621 1998-07-09 1999-07-08 Preparations de cellules epitheliales humaines pures et viables a partir de tissu digestif Ceased WO2000003002A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU45960/99A AU4596099A (en) 1998-07-09 1999-07-08 Pure and viable human epithelial cell preparations from digestive tissue

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CA 2242680 CA2242680A1 (fr) 1998-07-09 1998-07-09 Preparations pures et viables de cellules epitheliales humaines a partir du tissu intestinal, et leur mode d'obtention
CA2,242,680 1998-07-09

Publications (2)

Publication Number Publication Date
WO2000003002A2 true WO2000003002A2 (fr) 2000-01-20
WO2000003002A3 WO2000003002A3 (fr) 2000-08-24

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PCT/CA1999/000621 Ceased WO2000003002A2 (fr) 1998-07-09 1999-07-08 Preparations de cellules epitheliales humaines pures et viables a partir de tissu digestif

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AU (1) AU4596099A (fr)
CA (1) CA2242680A1 (fr)
WO (1) WO2000003002A2 (fr)

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WO2008132722A1 (fr) * 2007-04-26 2008-11-06 Ramot At Tel-Aviv University Ltd. Cellules souches autologues pluripotentes de muqueuse buccale et procédé d'utilisation
US9717761B2 (en) 2011-11-21 2017-08-01 Ramot At Tel-Aviv University Ltd. Stem cell-derived neural cells for cell therapy in neurological disorders
CN117503800A (zh) * 2024-01-04 2024-02-06 北京益华生物科技有限公司 一种胃黏膜上皮细胞提取液及其制备方法与应用

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CN115161282A (zh) * 2022-07-22 2022-10-11 邓超 一种小鼠脑微血管内皮细胞与周细胞联合提取培养方法
CN118584107B (zh) * 2024-07-30 2024-10-15 四川省肿瘤医院 细胞角蛋白18在非疾病诊断治疗目的中作为小肠潘氏细胞和杯状细胞共同标记物的应用

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FR2634784A1 (fr) * 1988-08-01 1990-02-02 Inst Nat Sante Rech Med Lignees de cellules epitheliales intestinales immortalisees, leurs procedes de preparation et leurs applications en tant que systeme de production de facteurs de croissance et en tant que modele d'etude de la physiopathologie digestive fonctionnelle et cancereuse

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WO2008132722A1 (fr) * 2007-04-26 2008-11-06 Ramot At Tel-Aviv University Ltd. Cellules souches autologues pluripotentes de muqueuse buccale et procédé d'utilisation
US9534201B2 (en) 2007-04-26 2017-01-03 Ramot At Tel-Aviv University Ltd. Culture of pluripotent autologous stem cells from oral mucosa
US10570369B2 (en) 2007-04-26 2020-02-25 Ramot At Tel-Aviv University Ltd. Pluripotent autologous stem cells from oral mucosa and methods of use
US9717761B2 (en) 2011-11-21 2017-08-01 Ramot At Tel-Aviv University Ltd. Stem cell-derived neural cells for cell therapy in neurological disorders
CN117503800A (zh) * 2024-01-04 2024-02-06 北京益华生物科技有限公司 一种胃黏膜上皮细胞提取液及其制备方法与应用
CN117503800B (zh) * 2024-01-04 2024-04-05 北京益华生物科技有限公司 一种胃黏膜上皮细胞提取液及其制备方法与应用

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WO2000003002A3 (fr) 2000-08-24
CA2242680A1 (fr) 2000-01-09
AU4596099A (en) 2000-02-01

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