WO2000003002A2 - Pure and viable human epithelial cell preparations from digestive tissue - Google Patents

Abstract

The present invention relates to a method to separate a human digestive tissue epithelium from its underlying mesenchyme comprising the step of incubating the human digestive tissue with a 100 % MatrisperseTM solution for 8-20 hours to enable a separation of the epithelium from the mesenchyme. In particularly preferred embodiments of the invention, the digestive tissue is intestine or gastric tissue. The invention further relates to primary epithelial cell cultures derived from human digestive tissue which have been obtained by a non-enzymatic treatment of digestive tissue and which are pure, functional and viable in culture for at least two days in the absence of a mesenchymal support. In one particular embodiment, the intestinal epithelial cell culture comprises goblet cells and absorptive cells which have retained substantially all of the characteristics found in vivo in the intestinal epithelium from which the primary cell culture was derived. In another embodiment, the gastric epithelial cell culture comprises mucus secreting cells, parietal cells and a significant amount of zymogenic chief cells. In addition, the invention 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-like cell line which can be induced to differentiate, comparable to cells of the upper half of the crypt. The invention relates to the use of the primary cell cultures of the present invention as model systems to identify drugs and compounds which affect same.

Description

TITLE OF THE INVENTION

PURE AND VIABLE HUMAN EPITHELIAL CELL PREPARATIONS FROM DIGESTIVE TISSUE

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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]. Because of the unavailability of normal human intestinal epithelial cell lines, most of the knowledge about human intestinal cell regulation has been derived from studies conducted on cell cultures generated from experimental animals [15-19] or human colon cancers [13-14]. However, the limitations of these models are well documented. On one hand, it appears more and more evident that the regulation of gene expression during development and along the crypt-villus axis differs fundamentally between man and animal models [20,37-57] For instance, lamιnιn-1 , which is directly associated with intestinal cell differentiation in the human [13,117,39] is restricted to the proliferative compartment in rodents [40] This suggests that observations performed on experimental animal models cannot always be transposed to the human situation It follows that results obtained with such animal models cannot be assumed to be predictive of the situation in humans On the other hand, a number of human colon cancer cell lines have been used to investigate various aspects of intestinal cell function and regulation but the theoretical limitations with these models are obviously their cancerous nature and their colonic origin [13-14,20] Thus, once again, the transposition of results obtained using these cancer cell lines to the normal human situation is limited

Two recent progresses in the generation of human normal intestinal cell lines should help to overcome some of these limitations First, human intestinal cell lines with typical proliferative crypt cell characteristics are now available [44] Like their rodent counterparts [95,96] that are unable to undertake a differentiation program, these cell lines should improve the understanding of many of the intestinal, physiological and biological functions typical to man including cell-cell and cell-matrix interactions, cytokine and growth factor effects and metabolism Second, the production of conditionally immortalized human intestinal primary cultures with a temperature-sensitive SV40 large T antigen has been recently described [97] At 32°C, these cells proliferate and display crypt cell markers but a shift to 39°C results in an irreversible growth arrest and acquisition of an enterocyte- ke phenotype Thus, this system seems particularly well-suited for studying early events in cell differentiation in vitro, one that would be comparable to the process taking place in the intestinal crypt in vivo [97] However, in vitro models of fully differentiated normal enterocytes like those found on the villus are still lacking Because of their non-pro ferative status, normal fully differentiated enterocytes can only be maintained in vitro as primary cultures Until now, the usefulness of these cultures for experimentation has been limited by low recoveries, fibroblast contamination, and the fact that the cells can only survive and maintain a differentiated phenotype for very limited times, usually 1-2 days [44,97', 98], unless a mesenchymal support is provided [98] A possible explanation for this phenomenon is that permanent cellular damage occurs rapidly when epithelial cells are separated from their extracellular matrix support by using conventional dissociation methods [99,100]

There therefore remains a need to provide primary cultures of fully differentiated and pure human intestinal epithelial cells which can be maintained in culture for more than 2 days There also remains a need to provide such cultures which can be maintained in culture in the absence of a mesenchymal support Furthermore, there remains a need to provide a simple method to prepare such primary cell cultures which enables the obtention of cells which are representative of their normal counterparts in vivo Epithelial and mesenchymal tissues are separated by a specialized sheet-like extracellular matrix, the basement membrane (BM) BM components have received great attention as potential key elements for the mediation of epithelial-mesenchymal interactions involved in the regulation of intestinal cell functions [6-8], based on the observation that they are in direct contact with epithelial cells and that the molecules that form them have been identified as dynamic effectors of cell adhesion, migration and tissue-specific gene expression [9-12] The human intestinal BM has been found to contain most of the major components specific to BMs, namely type IV collagens, lammins and proteoglycans as well as a number of BM-associated molecules such as fibronectin and tenascin-C A number of specific cell receptors for these molecules, namely those of the integπn family, have also been identified [7] Furthermore, direct evidence that cell interactions with extracellular matrix molecules can control specific functions in the enterocyte has recently begun to accumulate Despite the importance of BM components for enterocyte regulation, little is known about the tissular origin of the molecules that compose it in the human intestine Indeed, in contrast to other organs such as the skin, where the origin of the various BM component mRNAs has been directly determined after separation of the epithelium and mesenchyme [16], the intestine has been proven difficult to dissect into pure epithelial and mesenchymal fractions The same can also be said of gastric tissue Determining the tissular origin of BM molecules in the developing digestive tissue such as intestine and gastric tissue appears to be of prime importance to understanding the role of these molecules in epithelial-mesenchymal interactions For instance, in situ hybπdization has revealed that the α1 (IV) chain of collagen originates mainly, if not exclusively, from the mesenchymal compartment in the rodent small intestine [17] pointing out the requirement of the mesenchyme for the formation of a complete epithelial BM in this organ [7] Unfortunately, in situ hybridization has been proven difficult to apply to the detection of transcripts expressed at relatively low levels such as several of those encoding BM molecules [18 19] Therefore, up to now, most of the knowledge about the tissular origin of extracellular matrix molecules localized at the epithelial-mesenchymal interface results from various approaches including co-culture systems and interspecies hybrid tissue recombination [6,7] Although these approaches have significantly contributed to the establishment of the concept that the intestinal epithelial BM is composed of molecules produced by both cell types, the use of tissues obtained from laboratory animals and/or cancer cell lines in these studies precludes a direct extrapolation of their results to the normal human situation Once again, therefore, there remains a need to provide human epithelial cell cultures which are representative of their in vivo counterparts such that fundamental and practical questions can be answered and adaptable to the in vivo situation

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 tπglycerides 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 renewal and differentiation of the gastric epithelium is therefore a complex process which is likely to be regulated by a number of factors including growth factors, hormones, cytokines, and cell-cell and cell- matrix interactions [56, 57-62] In order to understand the specific implications and the underlying mechanisms of action of these modulators, adequate cellular models must be available Because of unavailability of normal gastric epithelial cell lines, most studies focused on cell cultures generated from animals [63-66] and human gastric cancers [61 , 62] When cells are isolated using a scraping technique, mixed gastric epithelial cells are rapidly overgrown during the first two days of culture by dividing mucous cells [67, 68] On the other hand, the enzymatic approach which commonly uses collagenase, pronase and hyaluronidase, generates cultures that are mainly composed of mucus-secreting cells, with a low proportion of parietal cells and absence of chief cells [69,70] The dissociation procedure yields a suspension of dispersed cells that necessarily require the presence of a biological substratum for their attachment and survival [71 ,72] In most if not all cases, the primary culture systems prepared using the above techniques are not representative of the intact gastric epithelium, as examplified by a systematic absence of chief cells, the zymogenic cell type involved in the gastric digestion of dietary proteins and tnglycerides In the past, several attempts have been made in order to generate epithelial monolayer cultures from stomach of various animal models

[63,64,68,70] and adult human stomach [67] Conventional techniques for isolating gastric epithelial cells mostly included the use of dissociating enzymes such as collagenase, dispase or pronase [63-66] These protocols resulted in poor epithelial cell recovery and often some degree of contamination by mesenchymal cells Furthermore, these gastric primary cultures consisted almost entirely of surface mucous cells with very few parietal cells and, most notably, no chief cells [64 71 ,84] Thus, there remains a need to provide an in vitro model of gastric epithelium which is representative of the intact gastric epithelium There also remains a need to generate gastric cell cultures that have a higher proportion of parietal cells and chief cells than that of the prior art

There also remains a need to provide gastric cell cultures which do not require the presence of a substratum for their attachment and survival and/or which can remain viable longer than the cultures of the prior art

There thus remains a need to provide a model human cell system of digestive epithelium that enables a direct extrapolation to the normal human situation There also remains a need to provide a method to purify and isolate epithelial cells from human mesenchymal digestive tissue Further, there remains a need to provide an in vitro model system of human digestive epithelial cells which are pure viable and functional

The present invention seeks to meet these and other needs The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety

SUMMARY OF THE INVENTION

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

In addition, the present invention relates to an in vitro model system of digestive epithelial cells which are pure, viable and functional More specifically, the invention relates to human intestinal epithelial primary cultures which are substantially free of mesenchymal tissue, are viable in culture for about two weeks and are representative of normal human intestinal epithelium

More specifically, the present invention relates to a simple procedure that allows an efficient dissociation of human digestive tissue into pure epithelial and corresponding mesenchymal fractions In one particular embodiment, 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 present invention also relates to a simple procedure to isolate the villus epithelium from the fetal human small intestine This approach, based on the use of Matnsperse™, a non-enzymatic solution initially designed to isolate epithelial cells grown on Engelbreth-Holm-Swarm (EHS) biomatπx, has allowed the dissociation of the integral villus epithelial lining from its underlying mesenchyme, with a high degree of purity, as assessed with different markers Furthermore, these fully differentiated intestinal cells can be maintained in culture for up to 10-12 days without having to provide a mesenchymal support, when plated on a thin coating of type I collagen

In another embodiment of the present invention, 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 gastπc epithelium can be easily and efficiently isolated from the developing human gastric mucosa with Matnsperse™ The resulting preparations consisting of intact epithelial cell sheets or large aggregates can be maintained as pπmary 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 The use of Matnsperse™, 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 Moreover 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-matrix interactions and apoptosis

Before the present invention, no non-enzymatic method to separate the human digestive epithelium from its underlying mesenchyme was known Furthermore, no such method was known that yielded an epithelial cell preparation that could grow for up to 10 days 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 More specifically, the method of purification and isolation of epithelium from human digestive tissue comprises an incubation of human digestive tissue with Matnsperse™ 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 In accordance with the present invention, there is thus provided 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 Matnsperse™ for a sufficient time to enable a separation of the epithelium from the mesenchyme In accordance with the present invention, there is also provided a primary cell culture of fully differentiated and pure human digestive epithelial cells which can be maintained viable and functional in culture for more than 2 days

In addition, in accordance with the present invention, there is provided an in vitro cell model system of a normal crypt-villus axis of human intestine comprising a pπmary 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

Further, in accordance with the present invention, there is provided a method of generating functional chief cells from human fetal stomach, comprising an incubation of the human fetal stomach with an effective amount of Matnsperse™ and for a sufficient time to enable an obtention of the functional chief cells in a primary cell culture of gastric epithelium.

In accordance with the present invention, there is provided, in addition, a method of preparing primary epithelial cells from human digestive tissue, comprising an incubation of the human digestive tissue with an effective amount of Matnsperse™ 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 As used herein, the terminologies "normal cells counterpart",

"intact cells" or the like, 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 It will be clear to the person of ordinary skill that the 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) As examplified hereinbelow, the 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 Thus, the person of ordinary skill will be able to adapt the incubation time and concentration of Matnsperse to meet particular needs

It will be recognized that 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

As used herein, the terms "molecule" or "compound" broadly refer to natural, synthetic or semi-synthetic molecules or compounds The term "molecule" therefore denotes for example chemicals, macromolecules, cell or tissue extracts (from plants or animals) and the like Non limiting examples of molecules include nucleic acid molecules, peptides, antibodies, carbohydrates and pharmaceutical agents The agents can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modelling methods such as computer modelling The terms "rationally selected" or "rationally designed" are meant to define compounds which have been chosen based on the configuration of the interaction domains of the present invention As will be understood by the person of ordinary skill, macromolecules having non-naturally occurring modifications are also within the scope of the term "molecule" For example, peptidomimetics, well known in the pharmaceutical industry and generally referred to as peptide analogs can be generated by modelling as mentioned above The primary cell cultures in accordance with the teachings of the present invention find utility in identifying compounds or molecules which can affect their proliferation, metabolism, differentiation transport, homeastasy and the like

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the invention, reference will now be made to the accompanying drawings, showing by way of illustration a preferred embodiment thereof, and in which 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 Matnsperse™ 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)

Figure 2 shows the western blot analysis of intact small intestine and Matπsperse-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-cadheπn (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 α-smooth muscle actin (αSMA) were present only in the intact and the mesenchymal fraction

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 α1 (IV) collagen chain (C α1 (IV)), laminin β1 chain (Ln β1), fibronectin (Fn), decoπn (Dc) SPARC/osteonectin (SPARC), E-cadheπn (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 α1 (Ln α1) and α2 (Ln α2) chains collagen α5(IV) (C α5(IV)) and α6(IV) (C 6(IV)) chains, tenascin-C (Tn-C), E-cadheπn (E-cad), sucrase-isomaltase (SI) and S14

Figure 5 shows the dissociation kinetics of epithelium from explants of human fetal stomach during MATRISPERSE™ treatment A) Representative light micrographs of intact gastric explant ( 7 weeks of gestation showing the epithelium and the surrounding lamina propria Filled arrow indicate the base of forming glands (x20) B) Explant treated for 10 hours (mid- treatment) Blank arrows illustrate the initial detachment of surface epithelium in large fragments or intact sheets (x20) C) Explant treated for 18 hours Note the complete dissociation of the epithelium from the remaining underlying mesenchyme (x40)

Figure 6 shows the phase-contrast morphology of pπmary 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) ³H- 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 keratιn-18 (A), vimentin (B), ZO-1 (C) and E-cadheπn (D) Gastric epithelial cells are found positive for keratιn-18 while vimentin was detected only in some cells at the periphery of colonies Tight-junction and zonula adherens are localized homogeneously at the cell-ceil interfaces (Original magnifications A, B, and D, x40, C, x20)

Figure 9 shows the immunodetection of growth factor receptors (1 5 day of culture) A) Human EGF-R B) IGF1-R C) HGF-R or c- met protein 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 A) PAS staining showing intense reactivity in a majority (50-60%) of cells Dark staining at the center of the specimen reveals the presence of secreted mucus (x16) B) Bowie staining of pepsinogen-containmg cells with their dark violet appearing cytoplasm, as indicated by blank arrows (x16) Insert illustrates a positive cells at higher magnification (x40) C) Indirect immunofluorescence of H7K⁺-ATPase revealing parietal cells (x40)

Figure 11 shows the immunodetection of human pepsinogen (Pg5) in gastric ceils 1 5 day after plating A) A subpopulation of cultured cells shows an intense granular staining (filled arrows) (x40) B) Representative Western blot analysis of cultured cells at 1 5, 3 and 5 days showing Pg5 and keratιn-18 reactivity Densitometπc analysis illustrates the relative levels of Pg5 protein expressed in arbitrary units (AU) as the values were normalized to keratιn-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 showing HGL and keratιn-18 Densitometπc analysis show the relative levels of HGL protein expressed as arbitrary units (AU) as the values were normalized to keratιn-18 signals Values represent the mean ± SEM of 4 separate and independent experiments

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)

Figure 14 shows the effect of EGF on Pg5 and HGL activity and on HGL-mRNA expression A) Pg5 and HGL activity determined in three separate cultures (stacked bars) in absence (control) or presence of 100ng EGF/ml for 1 5 day of culture For each culture, the variation of activity is illustrated as the percentage of increase or decrease relative to its own control value B) Northern Blot experiments of HGL-mRNA performed in cultured cells after 1 5 day of culture The addition of 100ng/ml of EGF significantly reduces HGL transcripts (E) as opposed to controls (C) Densitometπc 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 Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments with reference to the accompanying drawing which is exemplary and should not be interpreted as limiting the scope of the present invention

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Herein is described a new procedure to separate the human digestive epithelium from its underlying mesenchyme based on the use of Matnsperse™, a dissociating solution initially designed to recover cells grown on extracellular matrix The human digestive epithelium thereby prepared is substantially pure, viable and provides a model system for the analysis of normal human digestive epithelium

Indeed, it is herein shown that fully differentiated human intestinal cells can be isolated from the fetal small intestine with a high degree of purity and maintained without visible alteration under minimal conditions in primary culture for up to 10 days The use of Matnsperse™, a commercial non- enzymatic preparation initially designed to isolate epithelial cells grown on EHS biomatπx, appeared to be instrumental in both the efficiency of the isolation procedure and cell viability preservation Of note, up to now, 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] Using Matnsperse™ 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 Western blot analysis The only other treatment known to achieve comparable "en bloc" isolation of the human intestinal epithelium was based on the use of chelating agents [108,109] However, intestinal cells isolated with chelators failed to survive in culture [110] Thus, while the active dissociating agent(s) in Matnsperse™ remaιn(s) unknown, they likely dιffer(s) from those used previously

Isolated intestinal epithelial fractions with Matnsperse™ were found particularly well suited for primary culture Indeed, a large proportion of isolated epithelial fragments adhered to the dish surface, particularly when precoated with collagen I, and began to spread, forming epithelial colonies that reached confluence within a few days At 6-7 days after the plating, the morphological and immunological analyses of the monolayer showed that these epithelial cells consisted of absorptive and goblet ceils and that both cell types retained most, if not all, of the characteristics found in the villus epithelium including well-organized brush borders, junctional complexes, functional markers such as sucrase-isomaltase and lactase-phlorizin hydrolase, and poor proliferative potential [57,109 110] Furthermore, these primary cultures of differentiated enterocytes (PCDE) can be maintained for up to 10 days without alteration These observations were relatively surprising in the light of the apparent mesenchymal support requirement for the maintenance of a differentiated phenotype in intestinal epithelial cells in culture [15,19,98,110] One key element could be the preservation of the tissue integrity achieved by Matnsperse™ treatment Indeed, morphologically, the epithelial layer appeared particularly well preserved during the isolation procedure allowing, after the initial adhesion, epithelial cells to gradually migrate onto the surface of the dish during the first 3-4 days Another element may be related to the recent observation that permanent cellular damage occurs rapidly when celis are separated from their extracellular matrix support [99,100], a phenomenon susceptible to occurrence when performing enzymatic-based protocols of epithelial-mesenchymal dissociation

These characteristics make PCDE a unique in vitro system of villus-hke intestinal cell culture Interestingly, based on their morphological and functional properties 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] Used in conjunction with the two recently developed crypt-hke cell lines HIEC and tsHFI [44,97], 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

Because they are relatively easy to generate in a reproducible manner, 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 For instance, the non-proliferating status of PCDE cells gives this model a great interest for studying intestinal restitution, a repair process by which epithelial continuity is rapidly re-established after various forms of injury by migration of viable villus epithelial cells from areas adjacent to the injured surface to cover the denuded area Since this process does not require cell proliferation, 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 Up to now, the knowledge of the origin of epithelial basement membrane components in the intestine comes mainly from experiments performed on laboratory animals [17,33-36] However, it has become evident that results from experimental animals cannot always be directly extrapolated to the human Indeed, 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] For instance, 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] These systems have provided valuable information on the cellular origin of BM molecules but the use of adenocarcinoma cell lines such as Caco-2 and HT29 as the epithelial component has been at least theoretically, their main drawback A new procedure to separate the human intestinal epithelium from its underlying mesenchyme based on the use of Matnsperse™, a dissociating solution initially designed to recover cells grown on extracellular matrix, is shown This approach has allowed, for the first time, the possibility to investigate the respective epithelial and mesenchymal contributions to the BM in the normal human intestine

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 The use of Matnsperse™, a non-enzymatic dissociating solution, allowed the isolation of all epithelial cell types found in gastric epithelium and their maintenance in primary culture for up to two weeks 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

The present invention is illustrated in further detail by the following non-limiting examples

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 musculaπs propria 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 Matnsperse™ solution (Collaborative Biomedical Products, Becton Dickenson Labware, Mississauga, Ontario, Canada) and incubated at 4°C for about 8-1 Oh without agitation Then, flasks were gently shaken to separate the epithelium The remaining fragments, free of epithelial lining, were recovered and washed twice in PBS The epithelial suspension was also washed twice in PBS (100xg, 7mιn), resuspended in Dulbecco's modified Eagle's medium (DMEM, Gibco/BRL) supplemented with 4 mM glutamine, 20 mM Hepes, 50 U/ml penicillin, 50 μg/ml streptomycin, 5 ng/ml recombinant human epidermal growth factor (all obtained from Gibco/BRL), 0 2 lU/ml insulin (Connaught Novo Laboratories, Willowdale, Ontario, Canada), and 5% fetal bovine serum (FBS, CELLect Gold, INC/Flow, Costa Mesa, CA), plated in 2-3 60mm culture dishes (Falcon, Becton Dickenson Labware Mississauga, Ontario Canada) and/or 8-well Lab-Tek slides (Nunc Inter-Med, Rockilde, Danmark) previously coated or not with a solution of 50 μg/cm² rat tail collagen I (Collaborative Biomedical Products, Becton Dickenson Labware, Missisauga, Ontario, Canada) and cultured at 37°C with 5% CO₂ For each preparation, complete dissociation of the epithelium from the mesenchyme and lack of crosscontamination were confirmed at the protein level with tissue- specific markers [21] Total RNAs from intact tissues and mesenchymal and epithelial fractions from either small intestine or colon were prepared by the guanidine isothiocyanate-phenol method [22] EXAMPLE 2 Northern hybridization analysis

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 μg) was confirmed by hybridization to a rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene control Prehybπdization and hybridization were performed as described previously [18]

Hybridizations were performed with the followed random primed ³ P-labeled cDNA probes (Multipπme™ 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 decoπn fragment (Gibco BRL) and a 2 2 kb BamH I pig SPARC fragment (kindly provided by Dr J Sodek) The E-cadheπn 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

EXAMPLE 3

Reverse-transcriptase PCR

Conditions for amplification of E-cadheπn, sucrase-isomaltase and S14, used as endogenous control, have been described previously [29,30] For the laminin α1 chain, the sense primer 5'-aagtggcacacggtcaagac-3' and the antisense primer 5'-gacaagagctgcatatccgc-3' [31] were used For the laminin α2 chain, the sense primer 5'-tcaaagtatctgtgtcttcagga-3' and the antisense primer 5'-ccagtgaatgtaatcacacgtacagc-3' [32] were used

For tenascin-C, 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 hybπdyze 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 μM of both primers for 28 cycles of denaturation (1 mιn 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 μM dNTP's and 2 5 U of Taq polymerase (Roche Branchburg, NJ)

EXAMPLE 4 Sampling of gastric tissue specimens, cell isolation and culture

Tissues from 48 fetuses varying in age from 17 to 20 weeks of gestation (postfertihzation ages were estimated according to Streeter [73] 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 pyloπc segments were removed from the stomach leaving the body and fundic regions Tissue specimens were cut into explants (3x3mm²) 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 Matπgel™ as previously applied to human fetal small intestine (see above and [75]) Explants were immersed in ice-cold Matnsperse™ (from Collaborative Biomedicals, Bedford, MA) for 16 to 20 hours and gently agitated for approximately 1 hour (4°C) allowing dissociation of gastric cells as intact epithelial sheets or large aggregates The resulting material was centπfuged and resuspended in culture medium i e Dulbecco's Modification of Essential Medium (DMEM) and Ham F-12 formulation (1 1 ) supplemented with penicillin (50 U/mL), streptomycin (50 μg/mL) and 10% fetal bovine serum (v v, Cellect Gold FBS from ICN Pharmaceuticals Canada, Montreal, Quebec) Epithelial aggregates were fragmented mechanically into multicellular clumps, seeded in plastic 6-well multiwell plates (5x10⁴ cells in 3 mL) or 24-well plates (1 5x10⁴ cells in 1 mL), and left undisturbed for at least 24 hours to allow attachment Culture medium with non attached material was discarded and renewed every 48 hours

EXAMPLE 5 Growth and morphology of gastric cells

Cell cultures were characterized after selected time intervals using phase-contrast microscopy Cell number and viability were evaluated after each day by hematocytometer counting and Trypan blue exclusion technique performed on tryps -dissociated cells [76] The DNA synthesis rate was determined by incubating cells seeded in 24-well plates with 2 μCi/mL of [³H]- thymidine (Amersham Canada, Oakville, Ontario, spec act 80 Ci/mmol) during the last 12 hours of each time interval Total DNA was precipitated by tπchloroacetic acid treatment and then solubilized with 0 1 M NaOH + 2% Na₂C0₃ for 30 minutes and counted in a beta liquid scintillation system (Beckman Instruments, Fullerton, CA) Data were expressed as disintegrations per minute (DPM) per dish

EXAMPLE 6 Immunological methods Dye staining

Cell suspensions were seeded on sterile glass cover slips

(round-13 mm, from J B EM Services, Dorval, Quebec) At the end of culture intervals, specimens were rinsed with Hanks Balanced Salt Solution (HBSS) pH

7 4 and fixed in fresh 1 % formaldehyde diluted in 100 mM phosphate buffer pH 7 4 Periodic Acid Schiff (PAS) staining was performed to identify mucus- secreting cells and Bowie staining [77] for pepsinogen-containmg cells

(cytoplasm stained dark violet) and parietal cells (pink)

Antibodies

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/TGFα-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 Carπere, INSERM Marseille, France), 3) the anti- gastπc H7K⁺ATPase of parietal cells (1 800, Calbiochem, LaJolla, CA), 4) intermediate filaments epithelial keratιn-18 and mesenchymal vimentin (1 2000 and 1 200 respectively, Sigma-Aldπch Canada, Oakville, Ontario), and 5) junctional proteins cadheπn-E (1 800, Transduction Laboratories, Lexington, KT) and tight junction protein ZO-1 (1 500, Zymed Laboratories, San Francisco, CA) Appropriate secondary antibodies (anti-mouse, rabbit, guinea pig) conjugated to fluorescem isothiocyanate (FITC) were then added Specimens were examined on a Reichert Polyvar microscope equipped for epifluorescence and photo-graphed with Kodak TMAX film (400 ASA) Other antibodies which were used included the monoclonal HDPPIV-1 to human dipeptidylpeptidase IV (DPPIV), HAPN-1 to human aminopeptidase N (APN), HBB4/90 to goblet cell muc s [101], HSI-14 to human sucrase-isomaltase (SI) [102], mlac 1 to lactase- phioπzm hydrolase (LPH) [103], Caco-3/61 to the oncofetal crypt cell antigen (CCA) [104], CK-5 and CY90 directed to human cytokeratms 18 (CK-18, Sigma chemical Co , St-Louis, MO), Vim 3B4 to human vimentin (Vim, Boehπnger Mannheim), 1A4 to human smooth muscle actm ( -SMA, Sigma Chemical Co), HBBR-5 (Beauheu J -F et al , unpublished) and ZK-31 to human desmosome (Sigma Chemical Co ) Monoclonal antibodies directed to α-, β- and γ-catenιn were obtained from Transduction Laboratories (Bio/Can Scientific, Missisauga, Ont) A rabbit specific antisera directed to a crypt cell marker of 350 kD [105] was also used in this study

Indirect Immunofluorescence

Cells were grown on 8-well Lab-Tek slides (Nunc Inter-Med, Rockilde, Danmark) and washed twice with DMEM before use and fixed in ethanol (10 minutes, -20°C) for keratin 18 SI, DPPIV, APN, LPH, CCA, goblet cell, desmosome vimentin, E-cadheπn, α and β catenm or in 2% paraformaldehyde (45 mm 4°C) for HBBR-5, antι-ZO-1 and MIM 1/39 before immunostainmg as described previously [43,44] Primary antibodies were diluted 1 5-1 2000 (Ascites CK-5, 1 50 E-cadheπn, 1 1000, α-catenin, 1 500, β- catenin, 1 500, ZK-31 , 1 100, HBB 5/23, 1 100, mlad , 1 50, HBB 4/90, 1 100, sera antι-ZO-1 , 1 2000, MIM 1/39, 1 1000, Conditioned media HBBR-5, 1 2, HSI-14, 1 5, Vim 3B4 1 10,) in PBS containing 10% Blotto Secondary antibodies were fluorescem-conjugated goat anti-mouse and anti-rabbit IgG (Boehπnger Mannheim), used at a final dilution of 1 25 in PBS containing 10% Blotto In all cases, no fluorescent staining was observed when the primary antibody was omitted or replaced with species-matched non-imune serum. Western blotting

Cells seeded in 6-well plates were dissociated and lyzed as described [75] Proteins (80-100 μg aliquots) were separated by SDS-PAGE, transferred onto nitrocellulose membranes (BioRad, Mississauga, Ontario), incubated with primary antibodies against HGL, Pg5 and keratιn-18, and processed with the Western-Light Plus Chemilumi-nescent Detection System (Tropix, Bedford, MA) Autoradiograms exposed in a linear range were quantified by densitometπc analysis with a LKB XL Ultroscan (Pharmacia, Baie d'Urfe, Quebec) and signals were normalized to those of the keratιn-18 control EXAMPLE 7 Enzymic activities of the purified gastric epithelial cells

Cells were seeded in 6-well plates and enzyme activities were measured in absence or in presence of 100 ng EGF per mL as described previously for human fetal stomach [74] Briefly, lipolytic activity was measured using a long-chain tπglyceπde substrate (tπ [1- ⁴C]-oleaιc acid (Amersham, spec act 50 mCi/mmoi) Free [ ⁴C]-oleιc acid produced after a 1-hour incubation was separated by liquid-liquid partition in chloroform/methanol/heptane, precipitated with carbonate borate buffer pH 10 5, and quantitated by liquid scintillation spectrometry [78] Activity was expressed as nmoles of free fatty acids released per minute per milligram of protein Pepsin activity resulting from activation of pepsinogen at acid pH was measured by the method of Anson and Mirsky [79] using 1 mL of acid-denatured and dialyzed hemoglobin (Sigma-Aldπch Canada, Oakville, Ontario) as substrate and 100 μL of homogenate [79] Free ammo acid products generated by pepsin activity in the supernatant were quantitated by spectrometry (280 nm) using a tyrosine standard Pepsin specific activity was expressed in International Units (μmoles of substrate hydrolyzed per minute) per milligram of protein Protein was quantitated by the method of Lowry et al [80]

EXAMPLE 8 Northern Hybridization Analysis on gastric cells

Total gastric RNA used in Northern blot analysis was isolated as described [81] Equivalent amounts of RNA (20 μg) 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 densitometπc analysis, signals were normalized to those of the GAPDH control

EXAMPLE 9 Electron Microscopy

The preparation of samples for morphological and ultrastructural studies was described previously [106] Briefly, cultures were prefixed in situ for 30 mm at 4°C with a mixture of complete medium to which 2 8% glutaraldehyde in 200 mM sodium cacodylate (pH 7 2) was added in a 1 1 ratio (vol/vol) The samples were fixed for an additional 45 mm at 4°C in 2 8% glutaraldehyde in 200 mM sodium cacodylate (pH 7 2), and postfixed for 75 m (4°C) in 2% osmium tetroxide For transmission electron microscopy, samples were embedded in epoxy (LX-112 resin from Ladd, Burlington, VT), cut into small pieces that were re-embedded to perform transverse sections of the monolayer Ultrath sections were stained with uranyl acetate followed by lead citrate and analyzed on a JEOL JEM-100CX transmission electron microscope

EXAMPLE 10 Gel Electrophoresis and Immunoblotting Sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described previously [43,44] Total proteins from dissociated epithelia were obtained by resuspendmg pellets in sample buffer (2 3% SDS, 10% glycerol and 0 001% BPB in 62 5 mM Tπs- HCI, pH 6 8) containing 5% β-mercaptoethanol Total protein from isolated mesenchymes and intact tissues were obtained from homogenates in 2X sample buffer (4% SDS, 20% glycerol and 0 08% BPB in 125 mM Tπs-HCl, pH 6 8) containing 5% β-mercaptoethanol [43] All samples were boiled (105°C) for 5 mm , cleared by centrifugation (13,000 rpm, 10 mm, 20°C) and ahquoted for storage at -80°C Equal amounts of protein were loaded for each sample Separated proteins were transferred onto nitrocellulose membranes (ImmunoSelect, Gibco/BRL) according to Towbin et al (1979) Protein and molecular weight markers (42-200 kDa range, BioRad, Mississauga, Ont ,) were localized by staining with Ponceau Red Immunoblotting analyses were performed essentially according to the procedure already described [43,106] Briefly, membranes were blocked 3 hours at 37°C in PBS containing 10% Blotto and incubated overnight with either CY-90, HSI-14, E-cadheπn, Vim 3B4, SMA 1A4 or MIM 1/39 diluted 1 5-1 5000 (CY-90, 1 1000, HSI-14, 1 5, E-cadheπn, 1 5000, Vim 3B4, 1 200, 1A4, 1 500 MIM 1/39, 1 5000) in blocking solution After washing in PBS, membranes were incubated with alkaline phosphatase- conjugated goat anti-mouse or anti-rabbit IgG (BioRad) diluted 1 2000 in 10% Blotto-PBS then processed for alkaline phosphatase detection according to the instructions of the manufacturer (BioRad)

EXAMPLE 11 [³H]thymidine Incorporation

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 μCi/ml of [³H]thymιdιne was added and incubated for a further 12 h Cells were then washed twice and DNA was precipitated with two subsequent treatments of 10% tπchloroacetic acid After solubihzation with 0 3 M NaOH and neutralization with 1 5 N HCl, total [³H]thymιdιne incorporation was evaluated using a Beckman LS 6800 scintillation counter

EXAMPLE 12

Statistical analysis

Results for cell counts and biochemical data were reported as means ± SEM Statistical significance was fixed at 95% and was determined by analysis of variance followed by Student's t test when significance was indicated EXAMPLE 13

Dissociation of the intestinal epithelium from the mesenchyme using Matrisperse™

Dissociation of the intestinal epithelium from the mesenchyme was achieved by using Matrisperse™ 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 Matrisperse™ 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)

Isolated epithelial and remaining mesenchymal fractions were then analyzed for the expression of tissue-specific markers by Western blot As shown on Fig 2, intestinal epithelial cell markers such as sucrase-isomaltase (SI), E-cadheπn (E-cad), keratin 18 (K18) and the antigen MIM-1/39 were only detected in the intact small intestine (Fig 2, lane 1) and isolated epithelial fraction (Fig 2, lane 3) while the fibroblast cell marker vimentin and α-smooth muscle cell actm were both present in the intact intestine and corresponding mesenchyme (Fig 2, lanes 1 and 2 respectively) but absent from the epithelial fraction (Fig 2, lane 3) indicating that relatively pure epithelial and mesenchymal fractions can be obtained with this treatment

EXAMPLE 14

Cell viability of the isolated epithelial fractions

The cell viability of these isolated epithelial fractions was tested in vitro The epithelial fractions were resuspended in complete culture medium and plated onto culture dishes previously coated or not with rat tail collagen Under both conditions villus-like remnants adhered and the epithelium began to spread on the substrate over the first 24 h, forming islets of condensed epithelial cells (not shown) On plastic, the spreading slowly continued for an additional 24 h Thereafter colonies started to degenerate and gradually detach from the surface (not shown) On the contrary, on collagen, the spreading of the isolated epithehal-hke structures was maintained so that individual colonies continued to expand over the next 48-72h (not shown) until confluence was reached Surprisingly, at 5-6 days after plating, the cultures still appeared as typical monolayers and can be maintained as such for an additional 5 days before observing the first signs of degeneration (not shown)

EXAMPLE 15

Characterization of the epithelial cells cultures by electron microscopy and indirect immunofluorescence

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-cadheπn were all localized homogeneously at the cell-cell interfaces Finally, the expression of a number of intestinal cell markers was investigated Sucrase-isomaltase and ammopeptidase N were homogeneously expressed in the monolayer, while lactase-phlorizin hydrolase was found according to a mosaic pattern throughout the monolayer (not shown), an observation reminiscent of the in vivo situation (103,104).

EXAMPLE 16

Analysis of the proliferation potential of the epithelial cell cultures

Surprisingly, a crypt-cell marker, the MIM-1/39 antigen, was detected in a subpopulation of the monolayer according to a typical cytoplasmic granular staining. The proliferative potential of these cultures was then investigated for DNA synthesis 48 h after the plating, a stage when colonies are rapidly expanding. As shown in Table 1 , [³H] 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.

TABLE 1 DNA synthesis in PCDE and intestinal cell lines

Values are expressed as dpm/10³ cells and are mean + SE; number of cultures per group is indicated in parentheses. In all cases, cells were seeded in 60 mm diameter dishes and tested 48 h after the seeding. ^*Statistically significant difference between PCDE and HIEC-6 (p^0.02) or Caco-2/15 (p-c0.001). EXAMPLE 17

Complete dissociation of the epithelium from the mesenchyme using the intestine epithelial cell cultures

For each preparation complete dissociation of the epithelium from the mesenchyme was confirmed at the protein level (not shown) with tissue-specific markers including keratin 18 (epithelium), vimentin (mesenchymal cells) and α-smooth muscle actm (smooth muscle cells) However, assessing the tissular origin of extracellular matrix molecules required analysis at the transcript level By using a combination of Northern blot and RT-PCR analyses, we have tested the origin of a number of formal BM molecules such as laminin and the type IV collagen chains as well as several BM-associated molecules As shown in Fig 3, Northern blot analysis revealed that the α1 (IV) chain of collagen (C α1 (IV)) was of exclusive mesenchymal origin (lane 2) while the β1 chain of laminin (Ln β1) and fibronectin (Fn) were both detected in the epithelial and mesenchymal fractions (lanes 2 and 3) Transcripts for decoπn (Dc) and SPARC/osteonectιn/BM40 (SPARC) were only detected in the mesenchyme (lane 2) RT-PCR was used for the detection of transcripts expressed at relatively low levels As illustrated in Fig 4 cDNAs corresponding to the α1 and α2 chains of laminin (Ln α1 and Ln α2) as well as the α5(IV) and α6(IV) chains of collagen were amplified in both epithelial and mesenchymal fractions (lanes 2 and 3) On the other hand, tenascin-C (Tn-C) was exclusively detected in the mesenchymal fraction (lane 2) Similar observations were found from epithelial and mesenchymal fractions isolated from the colon (data not shown)

It is pertinent to note that Fn and the Ln α2 chain were both consistently found at a relatively lower level in the epithelium as compared to the mesenchyme in 4 different specimen (Fn 12% vs 88%, p< 001 , Ln α2 25% vs 75% p< 025) Finally, purity of epithelial and mesenchymal fractions used for these studies was confirmed at the transcript level as determined by the exclusive detection of E-cadheπn (E-cad) and sucrase-isomaltase (SI) in the epithelium (Figs. 3 and 4) and the restricted expression of Dc, SPARC and Tn-C in the mesenchyme.

EXAMPLE 18 Pure human intestinal epithelium cell cultures as a model system for normal human intestine

As summarized in Table 2, a number of molecules are localized at the epithelial-mesenchymal interface, namely formal BM components such as the laminins and type IV collagens but also BM-associated molecules such as fibronectin, tenascin-C and decorin [7].

TABLE 2

Epithelial and Mesenchymal Contribution to the Expression of Extracellular Matrix Components in Relation to Their Distribution in the Intestinal Mucosa

Distribution" Expression*

Upper Lower Cellular elements Musculariβ

Vill us-EMI crypt-EMI crypt-EMI Strom a of the LP mucosa Epithelium Mesenchyme

C α5<IV) + + + + + + + + + +

C 6(IV) + + + nd nd nd + + + +

Ln al + - - + + + + + +

Ln α-2 - -t- + - - - + + +

Ln l + + + - + + + + + +

Fn -t- + + + + + + + +

Dc - + + + + + - + +

SPARC - - - - + + - + +

Tn-C + -♦- + + + - + +

Note, nd, not detected. ^α Distribution of extracellular matrix molecules at the epithelial-mesenchymal interface (EMI) along the crypt-villus axis and in mesenchymal elements (stroma, cellular elements of the lamina propria (LP) and musculariβ mucosa) summarized from Ref. 7.

* Expression of extracellular matrix components determined in isolated intestinal epithelium and corresponding mesenchyme as illustrated in Figs. 1 and 2.

The analysis of their tissular origin has revealed interesting findings First, the exclusive mesenchymal origin of the α1(IV) chain of collagen in the human was confirmed, as previously observed in rodents [17,34] and in co-culture [43] is confirmed, while the α5(IV) and α6(IV) chains of collagen are of dual epithelial and mesenchymal origin suggesting that intestinal epithelial cells contribute to their BM-collagen network, by producing their own collagen, presumably as a [α5(IV)]₂α6(IV) complex Secondly, concerning laminins, lamιnιn-1 chains (α1 , β1 and γ1) have been shown to be of both epithelial and mesenchymal origin in the rat intestine [34,35] An epithelial origin for lamιnιn-1 was also suggested by its expression in intestinal Caco-2 cells The detection of laminin α1 and β1 chains in both compartments has confirmed the dual origin of lamιnιn-1 in the human intestine Lamιnιn-2 has a very restricted pattern of expression in the human intestine, being exclusively located at the lower portion of the crypts [39] Interestingly, the α2 chain of lamιnιn-2 was also found in both the epithelial and mesenchymal fractions, suggesting that crypt epithelial cells as well as their peπcryptai myofibroblasts contribute to lamιnιn-2 deposition

Among the BM-associated molecules tested herein, decoπn and tenascin-C, which are distributed predominantly at the epithelial- mesenchymal interface in the intact human small intestine [7], were found to be exclusively of mesenchymal origin, thus confirming previous observations performed with human in vitro models [41 ,43] Interestingly, 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) The possibility that 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 Herein, 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 β1 chain, suggests that the epithelial contribution of fibronectin at the epithelial-mesenchymal interface is rather minor in the intact tissue Finally, SPARC was exclusively detected in the mesenchyme SPARC/osteonectιn/BM40 has been found to be a BM component in some tissues [49,50] However, in the human intestine, it is exclusively distributed in the smooth musculature as determined by immunofluorescence (see Table 2) and the expression of its transcript has not been detected in the epithelium [51] SPARC thus served as a mesenchymal marker in conjunction with the epithelial markers SI and E-cad to validate the lack of cross-contamination of the epithelial and mesenchymal preparations Taken together, these data support the concept that the intestinal BM is constituted from components produced by both the epithelium and mesenchyme Based on their origins in the normal intact human intestine as determined herein and their well established distribution at the epithelial- mesenchymal interface [7], it is proposed that epithelial BM α5(IV)/α6(IV) collagen and lamιnιn-1 mainly originate from the epithelium while lamιnιn-2 and Fn are of dual epithelial and mesenchymal origin in contrast to α1 (IV)/α2(IV) collagen, Tn-C and Dc which are produced exclusively by the mesenchyme

EXAMPLE 19 Dissociation kinetics of gastric epithelium from explants of human fetal stomach

The complete dissociation of the gastric epithelium from the surrounding mesenchyme was achieved within 20 hours following Matrisperse™ treatment (Fig 5) In freshly dissected gastric explants, the epithelium lining the mesenchyme clearly extends from surface to base of gastric glands (Fig 5A) After 10 hours of incubation at 4°C in the dissociation solution, the epithelium begins to loosen and separates as intact epithelial sheets (Fig 5B) As the incubation period is carried out for 20 hours, the epithelium is completely dissociated leaving only the underlying mesenchyme (Fig 5C) EXAMPLE 20 Morphology and cell growth kinetics

Isolated epithelial sheets were mechanically fragmented and resuspended in culture medium Resulting cell aggregates were plated in culture dishes in the absence of biological matrix Following adhesion, cells began to spread forming dense epithelial colonies after 1 5 day of culture (Fig 6A) They reached confluency by day 3 and displayed a polyhedral epithelial morphology (Fig 6B) The integrity of the confluent monolayer was maintained until 7 days of culture (Fig 6C) and the first signs of degeneration (cytoplasmic vacuohzation) appeared in some cells around 14 days (Fig 6D) Cell counts increased by two folds after 2 days of culture after which they remained constant (Fig 7A) In parallel, incorporation of [³H]thymιdme into total DNA was determined during the last 12 hours of culture in each above intervals Increased incorporation of the radiolabeled precursor into DNA was noted after 1 5 day and peaked after 2 days of culture (Fig 7B) During the following days, [³H]-thymιdιne incorporation continues eventhough at a much lower rate

EXAMPLE 21 Characterization of epithelial cells The immunodetection of various markers confirmed the purity of epithelial cultures and their integrity All cells in growing colonies were positive for keratιn-18 (Fig 8A) and a very low number of cells expressing the mesenchymal cell marker vimentin were visualized only at their periphery (Fig 8B) The presence and cellular distribution of adherent junction components was also established Immunoflorescent staining for the ZO-1 protein associated with zonula occludens was homogeneously observed at cell-cell boundaries (Fig 8C) Similarly, the staining pattern for zonula adherens- associated E-cadheπn was seen at the peripheral margins of all cells (Fig 8D) The presence of specific growth factor receptors known to be normally expressed in fetal gastric epithelium was assessed using immunohistochemistry after 1 5 day of culture (Fig 9) All cells were positive for immunostaming of EGF (Fig 9A) and IGF-1 (Fig 9B) receptors in which membrane labeling was observed The presence of HGF receptor (c-met, Fig 9C) and KGF receptor (K- sam, Fig 9D) was visualized at plasma membrane and cytoplasmic domains of cells In order to ascertain the presence of different gastric epithelial cell types, histochemical analysis and immunodetection of specific gastric functional markers were performed (Fig 10) A positive PAS reactivity indicative of the intracellular accumulation of mucigemc granules was revealed in approximately 60% of total cells (Fig 10A) Moreover secreted mucus lying over the cells was also visualized A positive Bowie staining in a subpopulation of cells was evidenced as a violet cytoplasmic reaction indicative of the presence of pepsinogen and chief cells (Fig 10B) Furthermore, the presence of a small population (5%) of parietal cells was indicated by a positive pink reaction to Bowie staining and immunodetection of gastric H7K⁺-ATPase (Fig 10C)

EXAMPLE 22 Functional status of human gastric chief cells

In order to characterize the chief cell population positive to Bowie staining, indirect immunofluorescence was performed after 1 5 and 5 days of culture with 2 specific antibodies for known digestive enzymes characteristic of human chief cells namely pepsinogen and lipase As illustrated in Figs 11 and 12, anti-peps ogen (Pg5) and anti- pase (HGL) stainmgs were observed in a significant proportion of cells i e 20 to 25% after 1 5 day (Fig 11A, 12A) and 5 days (not shown) of culture Pg5 and HGL stainmgs were found associated with cytoplasmic secretory granules Western blotting experiments confirmed the cellular presence of the 34KDa Pg5 (Fig 11 B) and the 49KDa HGL proteins (Fig 12B) However, while the densitometπc determinations showed that Pg5 expression rose during the culture period, the expression of HGL decreased from 1 5 to 5 days of culture To further define the functional status of the chief cell population, enzymatic assays were performed at different culture periods Cumulative pepsin (Pg5) activity (cells + medium) showed a constant increase in primary culture, total activity was multiplied 2 5 times after 5 days compared to the beginning of culture (Fig 13A) However, total lipase (HGL) activity showed only a slight increase during the same time interval (Fig 13B) We therefore evaluated the capacity of chief cells to express and secrete both digestive enzymes between given culture intervals i e 1 5-3 days, 3-5 days As shown in Fig 13C, pepsin activity remained constant in the cells and their capacity to secrete pepsin in the culture medium was unchanged, allowing for the progressive increase of total activity seen in Fig 13A In contrast, eventhough HGL continues to be expressed during culture, it is rapidly secreted into the culture medium rather than accumulated in the cytoplasm of cells (Fig. 13D) Northern blotting was subsequently performed at the above selected culture intervals HGL-mRNA levels were high at the beginning of culture (1.5 day) but began to decrease onward (not shown) Since previous results from our laboratory have demonstrated that EGF reduces lipase activity as well as HGL- mRNA expression in organ culture [30, 32] The addition of 100ng/ml of EGF in primary culture resulted in a significant decrease of HGL cellular activity after 1 5 day of culture (Fig 14A) without affecting that of pepsin Furthermore, the presence of EGF also diminished HGL-mRNA levels (Fig 14B) during the same time interval This pattern of HGL-mRNA expression parallels that of lipase protein, indicating that lipase expression in primary culture is regulated at the transcriptional level

EXAMPLE 23 Pure human gastric epithelium cell cultures as a model system for intact human gastric tissue

The use of Matrisperse™ under the conditions formerly described for small intestine above [75] allowed the isolation of large gastric epithelial sheets almost devoid of mesenchymal contamination as assessed by indirect immunofluorescence of keratιn-18 and vimentin Following dissociation and mechanical fragmentation, cell clumps (10-30 cells) attached to the plastic dishes, spread and reached confluency within 3 days The ability of a subpopulation of cells to proliferate was confirmed by cell counts and incorporation of [³H]thymιdιne into DNA At confluency, the morphological and immunological analysis of the monolayer showed that these epithelial cells consisted of mucus, chief and parietal cells which retained most, if not all, of the characteristics found in the foveolus-gland axis including junctional complexes, specific growth factor receptors and functional markers [74, 82-84] Importantly, these primary cultures of gastric epithelial cells survived in the absence of added biological substratum for up to 9-10 days without visible alteration These observations were relatively surprising since the maintenance of a differentiated phenotype in existing gastric primary cultures required the support of mesenchymal constituents [68-70] This could be explained by the fact that single-dissociated epithelial cells show a poor capacity for survival in culture Another key element could be related to the recent observation that permanent cellular damage occurs rapidly when cells are separated from their extracellular matrix support [85,86], a phenomenon highly susceptible to occur when enzymatic based protocols of epithelial-mesenchymal dissociation are performed

As opposed to the existing gastric epithelial cell cultures [68,71 ,84], the Matπsperse™-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 Indeed, 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]) Herein, it was demonstrated that 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 cell culture appear similar to patterns previously reported with human fetal gastric explants maintained in organ culture [74] Furthermore, EGF which has been shown to downregulate HGL activity without affecting that of Pg5 in gastric explants [81 ,83] exerts the same modulatory action in human chief cells maintained in primary culture These data not only provide evidence of the functionality of chief cells within the primary cell culture but, by establishing that EGF exerts his modulatory role by acting directly on epithelial cells without the implication of the surrounding mesenchyme, illustrates the potential of such system

In summary, using the convenient and reproducible non- enzymatic dissociation technique presented herein, 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 integπn-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 application for this pπmary culture system would involve studying the pathogenesis and the mechanisms of action regarding Helicobacter pylori on human gastric epithelial functions in general and on digestive functions in particular [93,94].

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. Although the present invention has been described hereinabove by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

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Claims

I CLAIM:

1 A method to separate a human digestive tissue epithelium from its underlying mesenchyme compπsing the step of incubating said human digestive tissue with an effective amount of Matrisperse™ for a sufficient time to enable a separation of said epithelium from said mesenchyme

2 The method of claim 1 , wherein said human digestive tissue is human intestine

3 The method of claim 1 , wherein said human digestive tissue is gastric tissue

4 A pure viable and functional preparation of human digestive epithelium obtained in accordance with the method of claim 1 , 2 or 3

5 A primary cell culture of fully differentiated and pure human digestive epithelial cells which can be maintained viable and functional in culture for more than 2 days

6 The primary cell culture of claim 5, wherein said cells can be maintained viable and functional in culture from about 5 to about 10 days

7 The primary cell culture of claim 6, wherein said cells can be maintained viable and functional in culture for about 12 days 8 The primary cell culture of one of claim 5, 6 or 7, wherein said human digestive epithelial cells are representative of their normal counterparts in vivo

9 The primary cell culture of one of claim 5, 6, 7 or 8, wherein said human digestive epithelial cells are intestinal epithelial cells

10 The primary cell culture of claim 9, wherein said intestinal epithelial cells comprise goblet cells and absorptive cells which have retained substantially all of the characteristics found in vivo in the intestinal epithelium from which said primary cell culture was derived

11 The primary cell culture of claim 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10, wherein said culture can be maintained in culture in the absence of a mesenchymal support without a significant alteration of its biochemical or morphological markers

12 The pπmary cell culture of one of claim 5, 6, 7 or 8, wherein said human digestive epithelial cells are gastric epithelial cells

13 The primary cell culture of claim 12, wherein said culture comprises mucus secreting cells, paπetal cells and a significant amount of zymogenic chief cells

14 The primary cell culture of claim 12 or 13, wherein said primary cell culture can be maintained viable and functional for more than 2 days in culture in the absence of a biological substratum 15 The primary cell culture of one of claims 12, 13 or 14, wherein said culture is representative of the intact foveolus-gland axis of human stomach

16 Use of the primary cell culture of claim 12, 13, 14 or 15, as a model system for Helicobacter py/oπ-mduced disease

17 An in vitro cell model system of a normal crypt-villus axis of human intestine comprising a) a primary cell culture according to one of claim 9, 10 or

11 , b) a crypt-hke cell line comparable to undifferentiated proliferate cells of the lower crypt, and c) a crypt-hke cell line which can be induced to differentiate, comparable to cells of the upper half of the crypt

18 The in vitro cell model system of claim 17, wherein b) is

19 The in vitro cell model system of claim 17 or 18, wherein c) is cell line tsHFI

20 Use of the primary cell culture of claim 9, 10, 11 , 12, 13, 14 or 15, to test candidate drugs or compounds

21 A method of generating functional chief cells from human fetal stomach, comprising an incubation of said human fetal stomach with an effective amount of Matrisperse™ and for a sufficient time to enable an obtention of said functional chief cells in a primary cell culture of gastric epithelium

22 A method of preparing primary epithelial cells from human digestive tissue, comprising an incubation of said human digestive tissue with an effective amount of Matnsperse™ 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

23 The method of claim 22, wherein said human digestive tissue is intestine tissue

24 The method of claim 22, wherein said human digestive tissue is gastric tissue