KR20060021908A - Method for differentiating stem cells in cells that produce a pancreatic hormone - Google Patents

Abstract

The invention relates to a method for creating cells that produce a pancreatic hormone, said method involving the cultivation and differentiation of stem cells that have been obtained from the differentiated exocrine glandular tissue of an organism. The invention also relates to the use of the cells that produce said pancreatic hormone.

Description

METHODO FOR DIFFERENTIATING STEM CELLS IN CELLS THAT PRODUCE A PANCREATIC HORMONE}

The present invention relates to a process for differentiating stem cells into insulin-producing cells, which are cells producing pancreatic hormones, in particular, cells producing pancreatic hormones, a cell composition comprising cells producing pancreatic hormones and in particular produced by said process ( cellular composition), and in particular the pharmaceutical application of such cells and cell compositions.

It is known to differentiate stem cells into cells that produce insulin or other pancreatic hormones (see DE 102 90 025 T1, WO 02/059278). Conventional differentiation techniques have significant drawbacks as well as the complex process conditions that must be maintained for cell differentiation, as well as the need to bear in mind progenitor cells for cell differentiation. Conventional processes are actually based on differentiating cells from a collection of embryonic stem cells (so-called embryoid bodies). However, the large availability of embryos for cell differentiation is ruled out for several reasons, especially moral considerations. DE 102 90 025 T1 suggests the differentiation of adult stem cells. However, adult stem cells obtained by conventional processes only obtain the desired differentiation results in a limited range.

It is an object of the present invention to avoid the limitations associated with embryonic stem cell use and nevertheless provide an improved process for differentiating stem cells into cells producing at least one pancreatic hormone suitable for mass use under simple process conditions. The object of the present invention includes the achievement of an improved process for the generation of cells which produce pancreatic hormones to be implanted, especially in patients with pancreatic diseases, in particular diabetes.

This object is achieved by a process having the properties of claim 1, a cell having the properties of claim 22, and a cell composition having the properties of claim 24. Preferred embodiments and applications are derived from the dependent claims.

With regard to the process, the above-mentioned objects are achieved by culturing and differentiating non-embryonic stem cells extracted from tissues of the differentiated exocrine glands of the organism. A particular advantage of this process is that it produces cells that produce pancreatic hormones (hormone-producing cells) without differentiation from embryonic stem cells which are conventionally used. The inventors have found that adult stem cells isolated from exocrine glandular tissue are pluripotent, exhibit high division capacity and strong growth. This provides an effective source for differentiateable cells in which hormone-producing cells can be generated on a large scale.

The exocrine glandular tissue used in accordance with the present invention may be derived from adult, immature or non-human fetal organisms, preferably postnatal organisms. The concept of “adult” as used in this specification therefore refers to the developmental stage of the initial tissue, not the donor organism from which the tissue is derived. "Adult" stem cells are non-embryonic stem cells.

The exocrine gland tissue is preferably isolated from salivary glands, lacrimal glands, sebaceous glands, sweat glands, glands of the reproductive tract containing the prostate gland, or from gastrointestinal tissues containing the pancreas or from hepatic glands. In a more preferred embodiment, acinar tissue is involved. The glandular tissue is particularly preferably derived from the pancreas, parotid gland or submandibular gland.

A more important advantage of the process according to the invention is that the stem cells can be efficiently obtained from living donor organisms, for example from the salivary glands of humans or from human salivary glands, without the donor organism being substantially adversely affected. It is in fact. This is particularly desirable not only in terms of the possibility of further observing a donor organism associated with any disease, but also in moral terms.

According to a first embodiment of the present invention, stem cells isolated primarily from an organism are used as sources for further culture and differentiation into hormone-producing cells. This variant has the advantage that the process is particularly simple to execute. Desired differentiated cells can be obtained directly from the primary medium. In general, according to a modified embodiment of the invention it is observed that stem cells isolated from an organism first appear as a collection into so-called organoid bodies. This modification has the advantage that an effective reservoir for a significant amount of differentiated cells is made of organoid bodies. The inventors observed that stem cells isolated from exocrine glandular tissue form organoid bodies that exhibit strong growth into tissues having diameters of several millimeters or more when fed with nutrients.

According to a first variant of using an organoid body as a source for a desired hormone-producing cell, the present invention provides that stem cell differentiation occurs in the organoid body and the differentiated hormone-producing cell is subsequently separated from the organoid body. to provide. According to a second modification, stem cells are secondarily separated from the organoid body in an undifferentiated state, cultured and differentiated into desired hormone-producing cells. This example has the advantage that the organoid body is particularly simple to carry out the process because it exhibits spontaneous migration and growth of stem cells or differentiated cells in adhesion media available for further culture and differentiation.

The process according to the invention can be carried out in that the hormone-producing cells formed spontaneously from the primary or secondary isolated cells or from the organoid bodies are selected and further propagated. According to a preferred embodiment of the present invention the stimulation of the cell medium is promoted in the differentiation of hormone-producing cells. Stimulation has the advantage of increasing the efficiency and rate of desired hormone-producing cell development. According to a first variant, after the differentiation of stem cells into hormone-producing cells, their stimulated proliferation is promoted in the culture medium. According to a second variant, stimulation of stimulated differentiation of stem cells into the desired hormone-producing cells is promoted.

According to the present invention, the stimulus may comprise one or more of the following stimulus treatments, which may be performed simultaneously or sequentially. Treatment is coculture with the supernatant of the primary medium of the endocrine pancreas or of the cell line of the endocrine pancreas, co-cultured with differentiated cells of the endocrine pancreas or cell lines derived therefrom, fixed or provided in a liquid state. The melt can be designed to be treated (imprinted) with molecular differentiation factors or to activate genes in stem cells. In addition, stimulation can be carried out by adding already differentiated, hormone-producing cells and / or other substances, such as hormones, or cell types that affect differentiation.

If imprinting is performed with a fixed growth factor, a differentiation factor is preferably used that is immobilized on a movable carrier that can be placed relative to stem cells. This can preferably achieve intentional differentiation of individual stem cells or of specific stem cell populations. Carriers are, for example, synthetic materials that are beneficial for designing with differentiation factors, or biological cells in which differentiation factors are placed in the cellular membrane.

If, according to a more preferred embodiment of the present invention, identification and selection of differentiated cells from the cell medium is facilitated, this may result in further use of the generated pancreatic hormone producing cells. In particular, cell compositions consisting of all or most of the hormone-producing cells may be provided. If the selection is carried out with a known sorting method, such as a preparative cell sorting method or sorting in a fluidic microsystem, this may bring the advantage of being compatible with the biological procedures of conventional cells. .

Another advantage of identification and selection lies in the fact that cells not identified as hormone-producing cells and consequently not selected from the process culture can belong to further culture and differentiation. This may preferably increase the output of the process according to the invention.

According to another preferred variant of the invention, the stem cells are obtained from tissue derived from the gland of the secretory or gastrointestinal tract of an organism to generate hormone-producing cells. Stem cells are in particular isolated from tissues consisting of or including glandular tissue. Ingestion from the pancreas can bring advantages for the use of further tissue parts of the pancreas for said stimulation. Acquisition from salivary glands can result in treatment while protecting donor organisms.

Preferred donor organisms are vertebrates, especially mammals such as, for example, humans. When using human stem cells, the isolation of stem cells is carried out from non-embryonic states, i. In the case of nonhuman donor organisms it is also possible to use tissue differentiated in the fetal state.

Hormone-producing cells produced according to the invention are preferably used for therapeutic purposes. According to a first variant, treatment of humans with hormone-producing cells obtained from animal organisms can be facilitated. In addition, it is possible to treat humans with hormone-producing cells obtained from other humans. Particular preference is given to self-treatment of humans with hormone-producing cells obtained from their immature or adult stem cells. Treatment may be associated with pancreatic disease, metabolic syndrome or metabolic disease, in particular diabetes, hyperglycemia or worsening glucose tolerance. Insulin is produced by certain choices, such as pancreatic hormones.

One independent subject of the present invention is isolated cells producing differentiated pancreatic hormones from stem cells derived from the differentiated endocrine gland tissue of the organism. Such isolated cells are preferably obtained by the process according to the invention.

Another independent subject of the invention is a cell composition comprising the majority of such cells producing pancreatic hormones. The cell composition is preferably obtained by the process according to the invention. According to a preferred embodiment of the present invention, the cell composition may comprise other cells or substances, for example forming a matrix. Thus, another subject of the present invention is an artificial island of Langerhans.

According to a preferred embodiment of the invention the cell composition comprises an envelope or three-dimensional matrix in which hormone-producing cells and other cell types are arranged. The envelope or three-dimensional matrix consists of, for example, alginate, collagen, implantable material, polymer (biopolymer or synthetic polymer). Semipermeable materials are used that accept hormones (eg insulin) but do not allow cells to pass out. Other cell types include, for example, stem cells in pancreatic tissue and / or adjacent cells of Langerhans islet. Formation of the envelope (capsules) is particularly desirable because hormone-producing cells are fixed with them particularly efficiently and, if necessary, also prevented from further growth. The diameter of the capsule-cell composition or matrix-cell composition described herein is several hundred μm to several mm.

Another independent subject matter of the present invention are pharmaceutical compositions and pharmaceutical carrier materials having at least one hormone-producing cell or cell composition produced according to the present invention. All substances known in the pharmaceutical art for this function can be used as pharmaceutical, in particular organic carrier materials.

Other advantages and details of the present invention will be described with reference to the accompanying drawings in the following.

1 shows a flowchart of a differentiation process according to one embodiment of the invention.

2 shows a flowchart illustrating various modifications that provide stem cells.

3 shows a flowchart illustrating various modifications in which stem cells are aggregated into organoid bodies.

4 shows a procedure for producing an organoid body.

5 shows a picture of the culture of the organoid body.

6 and 7 show schematic illustrations of the imprinting of stem cells by molecular signaling factors.

8 shows a photograph showing insulin production of differentiated cells according to the present invention.

(i) Isolation of stem cells derived from differentiated exocrine glands of living organisms and aggregation into organoid bodies

Culture and differentiation according to the invention of stem cells obtained from differentiated exocrine glandular tissue of an organism comprises the steps described in FIG. First, isolation of stem cells from the organism (step 100) is performed to provide a source for stimulation and differentiation into hormone-producing cells. The details of separation and the various variants of the source for the next process step are described below. Stimulation of the cell medium used as a source (step 200) is carried out continuously within the framework of differentiation. After differentiation, identification and selection of hormone-producing cells is performed in step 300. Selected hormone-producing cells are collected and further prepared, if necessary, for example for pharmaceutical application (step 400). In addition, re-use of undifferentiated cells into the medium for the first time in order to be reaffected by stimulation and differentiation (step 500).

Other modifications that provide for further stimulation of undifferentiated stem cells are shown in FIGS. 2 and 3. According to FIG. 2, the stem cells are first isolated from the donor organism (step 110, see below). Starting from these primary isolated stem cells, a direct transition to stimulation (step 200) can be made according to the first variant (a). In general, aggregation of stem cells into organoid bodies is first performed in step 120. Details of the aggregation step are shown in FIGS. 3 and 4. Stem cells or already differentiated cells may be individualized and / or sorted from the organoid body in step 130 (step 140), while further stimulation of differentiation or proliferation of already differentiated cells may continue following modification (b). Can be.

According to FIG. 3, the aggregation of stem cells into organoid bodies according to step 120 includes the following partial steps. First, culture of stem cells in suspension (step 121, for example, see below) is performed. Alternatively, the culture can be carried out in agitated culture or in suspension without contact with the solid boundary surface in the electromagnetic cage. Aggregation into the primary organoid body is carried out in a three-dimensional suspension medium, either susceptible or equivalent (step 122). These organoids can be used as sources for individualization of cells. According to variant (a) in FIG. 4, a jump to step 130 in FIG. 2 takes place here. In general, the primary organoid body is first placed on a substrate in the culture medium for adhesive culture (step 123). Cell migration and layer formation take place on the substrate (step 124, see FIG. 5). As a result, after step 124, there are individualized stem cells or differentiated cells such that the jump to step 140 in FIG. 2 can occur immediately according to variant (b) in FIG.

Singularity of the organoid body in the adhesion medium is that the secondary organoid body is formed from the cell layer formed on the adhesion medium (step 125). The cells may be re-individualized from the secondary organoid bodies or the corresponding organoid bodies obtained according to the modification (c) in FIG. 3 (hop to step 130 in FIG. 2).

Individualization of the differentiated cells from the adhesion medium takes place according to known processes, for example, using marker properties for pancreatic hormones. For example, trypsinization of differentiated cells in adhesion medium and migration to their suspension is performed. Identification is carried out using a cell sorting device known, for example, with a magnetic bead to which antibodies bind only to hormonal-producing cells in a fluid microsystem, a preliminary cell sorting method, or a string. Surface markers are intentionally found and hormone-producing cells are selected with specific surface markers in a preliminary cell sorting process.

Identification of the cells found can also be performed on the basis of morphologically distinctive characteristics in which the hormone-producing cells are identified from undifferentiated cells or other cell types, for example. Morphological distinctive characteristics refer to, for example, the shape of a cell or nucleus array or the shape of granular components in a cell.

In addition, identification and selection can be carried out by means of cell electrophoresis analysis or analysis of other unique original properties such as surface mobility of hormone-producing cells.

In addition, stepwise identification and selection or mixed identification and selection from the above description can be achieved.

According to the symptom shown in FIG. 4, in order to obtain the cells, salivary tissues, preferably derived from salivary glands or pancreas, are brought into the medium mechanically and in granular form by enzymes (step 10 in FIG. 2). Bachem et al., Gastroenterol. 115: 421-432 (1998), Grosfils et al., Res. Comm. Chem. Pathol. Pharmacol. In contrast to the point of 79: 99-115 (1993), under the condition that the cell aggregates of Samsori remain intact to the maximum extent possible, no tissue block in which the cells are intended to grow is grown and rather tissue It is strongly subdivided.

These cells and cell aggregates are cultured in culture vessels for several weeks. The medium is changed every 2-3 days, at which time all differentiated cells are removed. The cells remaining in the medium are undifferentiated cells with unlimited dividing capacity.

Similar cells were isolated and described from the pancreas under the same conditions and designated in the form of myofibroblasts or stellate pancreatic cells (Bachem et al., 1998). However, in contrast to the cells according to the invention, infinite dividing capacity could not be observed. In addition, these cells could also be passaged an unlimited number of times without losing vitality.

In a second step 12 approximately 400-800 cells are each cultured in 20 μl medium in suspension. To do this, the drops are placed on the cover surface of the petri dish for bacterial culture and turned upside down on a Petri dish filled with medium so that the drops hang.

As a result of this type of culture, the organoid bodies, called organoid bodies, are formed within 48 hours and converted into suspension medium 16 for about 6 days. The partial view 18 in FIG. 4 shows the micrograph of such an organoid body 2.

The formation of the above mentioned secondary organoid bodies is illustrated in FIG. 5. First, the primary organoid body 2 forms a monolayer by migration or growth of cells 3 in a substrate of an adhesion medium, such as, for example, the bottom of the culture dish 20, and the secondary from the monolayer. The organoid body 4 grows after that. Further proliferation of cellular material is caused by incubating the primary organoid body 2 with the secondary organoid body 4. Hormone-producing cells can be obtained from primary or secondary organoid bodies (2), (4) respectively.

Another Representative Example of Isolation and Aggregation of Stem Cells

Isolation and aggregation of stem cells is described in detail in the following non-limiting examples.

Conventional general guidelines of action are maintained for processes for culturing biological cells and in particular mammalian cells. Even if there is no further explanation, the sterile environment is maintained in all cases during the process. The following buffers and media were used:

HEPES Stock Solution (pH 7.6) 2.383 g HEPES per 100 ml A. bidest. HEPES Eagle Badge (pH 7.4) 90ml modified eagle medium (MEM: 10ml HEPES stock solution Separation medium (pH 7.4) Digestion medium (pH 7.4) 32ml HEPES Eagle Medium bidest. 5% BSA 8ml 0.1M CaCl _{2 in} water 300 μl trasylol (200,000 KIE) 100 μl 20 ml separation medium 4 ml collagenase (Serva collagenase NB 8) Incubation badge Dulbecco's Modified Eagle's Medium (DMEM) Culture medium Dulbecco's Modified Eagle Medium (DMEM) DMEM + 4500 mg / l glucose + L-glutamine-pyruvate + 20% FCS (inactivated) + 1ml / 100ml penicillin / streptomycin (10000E / 10000μg / ml) or DMEM + 10% autologous Plasma + 1ml / 100ml penicillin / streptomycin heated to 37 ° C before use Differentiation medium (l) DMEM 380 ml FCS 95 ml 5 ml glutamine (GIBCO BRL) 5 ml (3.5 μl β-mercaptoethanol to 5 ml PBS) 5 ml non-essential amino acid (GIBCO BRL) 5 ml penicillin / streptomycin (GIBCO BRL) (10000E) / 10000μg / ml)

Instead of fetal bovine serum (FCS) in culture medium and differentiation medium, autologous plasma or less preferred but if desired autologous serum of tissue donors can also be used. This is particularly important when the tissue donor is identical to the recipient of the stem cell or the differentiated cell derived therefrom. Such self-treatment is desirable to avoid possible rejection.

The culture medium may also include other suitable basal medium known to culture eukaryotic cells, in particular mammalian cells, in place of DMEM medium, where differentiated cells die and the desired stem cells proliferate. Separation medium, incubation medium, differentiation medium may also include other conventional and suitable basal mediums.

Another differentiation medium that can be used for the stimulation provided according to the invention is the primary medium of the endocrine pancreas or the supernatant of the extracted cell line or the cell suspension with the differentiated or extracted cells of the endocrine pancreas. In particular, cells or populations of cells located in the endocrine tissue of the pancreas near the island of Langerhans can be used.

Examples 1 and 2 below describe in detail two working protocols for the isolation and culture of adult pluripotent stem cells from Samsori pancreatic tissue. Examples 1 and 2 relate to the isolation of stem cells from rats. Separation from other mammals, such as pigs, is carried out similarly. Example 3 describes a corresponding protocol for the separation of salivary glands from auricular tissue.

Example 1

1. Tissue Preparation and Cell Separation

10 ml of digestion medium is slowly and bubble-free injected into the pancreatic ducts of rats 2-3 years old with a syringe and a blunt cannula. The entire pancreas is thereby expanded and thus more easily detached and ready. The pancreas is then transferred to a glass beaker and another 5 ml of digestion medium is added. After removing the adipose tissue and lymph nodes, the tissue is very finely divided in a glass beaker with fine scissors, the adipose tissue floating on top is removed by suction, the suspension is then gasified with carbogen for 1 minute (if necessary) Repeatedly), wrapped in aluminum foil in a stirrer at 200 cycles / min and incubated at 37 ° C. for 20 minutes. The medium is then inhaled and carefully removed, the tissue is subdivided with scissors, the tissue fragments washed twice each with 10 ml of separation medium and 5 ml of digestion medium is added back to the tissue.

After gasification with carbogen again for about 1 minute and incubation at 37 ° C. for 15 minutes in an agitator at 200 cycles / min, the tissue pieces are divided by successive pull-outs with 10 ml, 5 ml, 2 ml, 1 ml glass pipettes and a single layer of filter Compress through the fibers. Cells individualized in this manner are now washed five times in incubation medium (37 ° C.), gasified with carbogen and centrifuged at 90 g each for 5 minutes. The pellets finally obtained are resuspended in incubation medium, gassed and dispensed into a tissue culture dish.

2. Culture of Cells

Tissue culture dishes with isolated cells are incubated in 37 ° C., 5% CO ₂ in an incubator. Medium is changed every 2-3 days and all differentiated cells are removed at this time.

On day 7 of culture, cells are passaged with a solution consisting of 2 mL PBS, 1 mL trypsin, 2 mL incubation medium. In this process, cells fall off the bottom of the petri dish. The cell suspension is centrifuged for 5 minutes, the supernatant is removed by aspiration, the cells are resuspended in 2 mL of incubation medium, transferred to a medium-sized cell media flask, and 10 mL of incubation medium is added. Medium is changed every 3 days.

On day 14 of culture, cells are passaged again, but with 6 ml of PBS, 3 ml of trypsin, and 6 ml of incubation medium. The cell suspension is centrifuged for 5 minutes, the supernatant is removed by suction, the cells are resuspended in 6 ml of incubation medium and transferred to three medium-sized cell media flasks, and 10 ml of incubation medium is added to each flask.

The cells are further cultured, passaged and seeded until the cells are in semi-fusion or fusion state.

Example 2

Pancreatic glands were obtained by anesthetizing male Sprague-Dawley rats (20-300 g) (CO ₂ ) and bleeding through the dorsal aorta. The cannula was introduced into the phloem across the duodenum and HEPES Eagle medium (pH 7.4), 0.1 mM HEPES buffer (pH 7.6), 70% (vol./vol.) Modified Eagle medium, 0.5% (vol./vol 1) (wt./vol.) Bovine serum albumin, 2.4 mM CaCl ₂ and collagenase (0.63 P / mg, Serva, Heidelberg, Germany). A 10 mL digestive medium containing was injected following the pancreas.

Before removing the pancreas, the attached adipose tissue, lymph nodes, and blood vessels were partially removed. Healthy pancreatic tissue is then placed in the digestive medium (at 20 ° C, lower metabolism), the pancreatic tissue is very finely divided by scissors, the adipose tissue floating at the top is removed by suction, and the tissue suspension is removed from the cell The gas was treated with Kabogen (Messer, Krefeld, Germany) without the medium (reduction of physical force) and with it the pH was adjusted to 7.4. The suspension was then incubated in 37 mL Erlenmeyer flask (covered with aluminum foil) with continuous stirring (150-200 times per minute) in 10 mL digestion medium. After 15-20 minutes, the floating fat and medium at the top were removed by inhalation and the tissue was re-divided and rinsed with medium without collagenase (repeat the process at least twice, preferably until the cell segment became clear). Then digestion medium was added and the mixture was again gasified with carbogen for about 1 minute. Digestion with collagenase was followed again at 37 ° C. for 15 minutes in the stirrer using the same buffer. After digestion, the samples were pulled out continuously by 10 mL, 5 mL, and 2 mL glass pipettes with narrow passages and separated, and the monolayer nylon nets (Polymon PES-200 / 45, Angst & Pfister AG, Zurich, Switzerland). The samples were centrifuged (37 ° C., 600-800 rpm in a Beckman GPR centrifuge, corresponding to about 90 g) 24.5 mM HEPES (pH 7.5), 96 mM NaCl, 6 mM KCl, 1 mM MgCl ₂ , 2.5 mM NaH ₂ PO ₄ , 0.5 mM CaCl ₂ , 11.5 mM glucose, 5 mM sodium pyruvate, 5 mM sodium glutamate, 5 mM sodium fumarate, 1% (vol / vol.), modified Eagle's medium, incubation medium containing 1% (wt./vol.) bovine serum albumin to further purify, equilibrate with carbogen and adjust pH to 7.4. The washing procedure (centrifugation, suction removal, resuspension) was repeated five times. Unless otherwise noted, the action was carried out at about 20 ° C. for the separation.

Samchully was resuspended in incubation medium and incubated at 37 ° C. in a humid atmosphere containing 5% CO ₂ . Sampling tissue died rapidly under these conditions (within 2 days), and dying differentiated cells fell away without damaging them from adjacent cells (protective separation), while non-dying stem cells sank to the bottom. Caught there, Differentiated sampling cells are not possible to do this. Most of the free floating samples and sample cells were removed at this time, incubation medium was seeded and replaced for the first time on the second or third day. At this point, the first stem cells and / or their precursors settled to the bottom and began to divide. The change of medium was then repeated every three days and differentiated Sampling pancreatic cells were removed each time the medium was changed.

On the 7th day of culture, cells were passaged with a solution consisting of 2 mL PBS, 1 mL trypsin (+ 0.05% EDTA), 2 mL incubation medium. The cells fell off the bottom of the culture dish. The cell suspension was centrifuged at about 1000 rpm (Beckmann GPR centrifuge) for 5 minutes, the supernatant was removed by suction and the cells were resuspended in 2 mL of incubation medium and transferred to a medium cell media flask and 10 mL of incubation medium. Added.

On day 14 of culture, cells were passaged again, this time with 6 mL PBS, 3 mL trypsin / EDTA, 6 mL incubation medium. The cell suspension was centrifuged at 1000 rpm for 5 minutes, the supernatant was removed by aspiration and the cells were also resuspended in 6 mL of incubation medium and transferred to three medium-sized cell media flasks and 10 mL of incubation medium was added to each.

On day 17, the third passage was conducted in a total of six medium-sized cell media flasks, and on day 24, the fourth passage was performed in a total of 12 medium-sized cell media flasks. Finally, all primary cells except stem cells were removed from the cell medium.

Stem cells can be further cultured, passaged and seeded as often as desired. Seeding is preferably performed at a density of 2 × 10 ⁵ to 4 × 10 ⁵ cells / cm ² in incubation medium.

Example 3

Isolation and culture of human parotid gland from exocrine tissue were performed similarly to the pancreatic protocol except for the following:

1. The exocrine tissue of the parotid gland was a mixture of glandular and coronary tissues.

2. Because salivary glands contain less proteases and amylases than the pancreas, it is possible to store salivary gland tissues for a while under refrigeration at about 4 ° C. before workup without overly damaging the tissues. As a specific example, the storage time was 15 hours and there was no negative effect on the isolation of the desired stem cells.

Example 4 below describes in detail the working protocol for producing organoid bodies.

Example 4

Undifferentiated cells are trypsinized with a solution of 10 ml PBS, 4 ml trypsin, 8 ml differentiation medium and centrifuged for 5 minutes. The resulting pellet is resuspended in differentiation medium in such a way that the dilution of 3000 cells per 100 μl medium is controlled. The cells take 3 ml pipettes and then suspend again.

The cover is removed from a petri dish for bacterial culture previously coated with 15 mL PBS (37 ° C.) per plate and the cover is inverted. Approximately 50 20 μl drops are placed on the cover using an automatic pipette. The cover is then quickly flipped over to allow the droplets to sag and placed in a Petri dish filled with differentiation medium. Petri dishes are then carefully placed in an incubator and incubated for 48 hours.

Next, the aggregated cells forming the organoid body in suspension are transferred from four covers to a petri dish for bacterial culture with 5 ml of incubation medium containing 20% FCS and further incubated for 96 hours.

Organoids are now carefully collected in a pipette and transferred to cell culture vessels containing differentiation medium and coated with 0.1% gelatin. In a particularly preferred embodiment of this process, a 6 cm Petri dish coated with 0.1% gelatin is used as a culture vessel, with 4 mL of differentiation medium placed before and then 6 organoid bodies in each. Chamber slides coated with 0.1% gelatin constituted another preferred culture vessel, in which 3 ml of differentiation medium was placed, followed by three to eight organoid bodies each. In addition, 24-well microtiter plates coated with 0.1% gelatin and 1.5 ml of differentiation medium per well, each containing four organoids, can also be used.

When cultured in this way, the ability of the cells to differentiate into organoid bodies is activated and can differentiate the cells, particularly hormone-producing cells. Cells can be stored and cultured as individual cells as well as organoid bodies, maintaining their pluripotency.

(ii) differentiation into organoids and / or tissues

After isolation and selective aggregation, stem cells or, optionally, already spontaneously differentiated hormone-producing cells are present in a particular cell medium or organoid body. In the following process, stimulated differentiation of stem cells or stimulated growth of already differentiated cells is performed according to step 200 (FIG. 1). To this end, one or more of the above-mentioned stimulation treatments are carried out. Stimulation of differentiation can generally be carried out with the above mentioned differentiation medium (l) in such a way that differentiation of stem cells or organoid bodies takes place. We found that differentiation yields, in addition to other cell types, in particular hormone-producing cells, which are then selected in the next process (step 300 in FIG. 1). In general, differentiation is provided with the differentiation medium mentioned above, with which the differentiation mainly takes place into hormone-producing cells and also the use of which simplifies the following identification and selection process. While the remaining examples relate to different differentiation medium and stimulation techniques, the following example 5 relates to stimulation with differentiation medium (l).

Example 5

For induction of differentiation, stem cells are preferably used after 42 days of culture. The use of cells stored for 12-18 months at the temperature of stem cells or liquid nitrogen after the third or fourth passage was also possible without problems.

First, cells were transferred to differentiation medium (l) with the above-mentioned composition, for example trypsin treatment of stem cell medium in culture medium, centrifugation at 1000 rpm for 5 minutes, pellets in differentiation medium (l) It was adjusted to a density of about 3 x 10 ⁴ cells / ml by resuspending and diluting to the required range.

Then, about 50 20 μl drops (600 cell counts / 20 μl) were placed inside the cover of the petri dish for bacterial culture (packed tips) and carefully flip the covers over the Petri dish filled with PBS so that the drops drooped. Let go. New tips were used for each cover. Petri dishes were then carefully placed in an incubator and incubated at 37 ° C. for 48 hours.

The cultivated cells, or organoids, were then transferred from each of the four covers to a petri dish for bacterial culture with 5 mL of incubation medium containing 20% FCS (hold the cover at an angle and holding about 2.5 mL). Rinse the organoid body with culture medium) incubated for another 5-9 days, preferably 96 hours.

Organoids were now carefully collected using a pipette and transferred to cell culture vessels coated with 0.1% gelatin and containing differentiation medium (l). Organoid bodies then grew and partially grew in individual cell regions that can be further individualized, expanded, and re-individualized. In a particularly preferred embodiment of this process, a 6 cm Petri dish coated with 0.1% gelatin was used as a culture vessel, in which 4 mL of differentiation medium (l) was placed and 6 organoid bodies each to it. Another preferred culture vessel is a chamber slide, coated with 0.1% gelatin and 3 mL of differentiation medium, each of which is then given three to eight organoids, a Thermanox plate for electron microscopy. (Nalge Nonc International, USA). Another alternative was a 24-well microtiter plate coated with 0.1% gelatin, with 1.5 ml of differentiation medium (l) per well followed by four organoid bodies in each.

In a preferred embodiment of this process, the organoid bodies were incubated for about 7 weeks in a 6 cm petri dish coated with gelatin and the individual organoid bodies were then microdissector (Eppendorf, Hamburg, Germany) according to the manufacturer's instructions. ) And then transferred to, for example, a new 6 cm Petri dish, chamber slide or thermox plate.

Example 6

Stimulation with the other differentiation medium mentioned above is carried out in a similar manner.

Example 7

6 illustrates the stimulation of the stem cells 1 in the substrate 21 by the molecular signal or the differentiation factor 5 included in the culture medium (step 200). Cell receptors 1a are located on the surface of stem cells 1. When a signal or differentiation factor is bound at the cell receptor 1a, the stem cells 1 are imprinted and differentiation into the desired hormone-producing cells begins. Molecular signals or differentiation factors (5) comprising known biological macromolecules, cellular components of pancreatic cells or cells, cellular components or cell populations, in particular located in the endocrine tissue of the pancreas near the island of Langerhans, are used.

Example 8

Fig. 7 thus illustrates the imprinting of stem cells 1 with already differentiated cells 6, where appropriate signals or differentiation factors are fixed or naturally present on the membrane surface.

Example 9

According to another variation, gene activation of stem cells can be promoted as described, for example, in DE 102 90 025 T1.

Example 10

8 shows insulin production of differentiated cells through micrographs of insulin-producing cells derived from human exocrine pancreas. The results were found with salivary glands of humans or other vertebrates. The points indicated by the arrows indicate the insulin produced by the cells. The white stripe corresponds to the original 20μm length.

The features of the invention shown in the foregoing specification, claims, and drawings may, in various embodiments thereof, be of particular importance as well as in combination for the practice of the invention.

It is an object of the present invention to avoid the limitations associated with embryonic stem cell use and nevertheless provide an improved process for differentiating stem cells into cells producing at least one pancreatic hormone suitable for mass use under simple process conditions. The object of the present invention includes the achievement of an improved process for the generation of cells which produce pancreatic hormones to be implanted, especially in patients with pancreatic diseases, in particular diabetes.

Claims (33)

A process for the generation of pancreatic hormone producing cells comprising culturing and differentiating stem cells obtained from differentiated exocrine glandular tissue of an organism. The process of claim 1, wherein the stem cells primarily isolated from the organism are cultured and differentiated. The process according to claim 1, wherein the stem cells are collected in an organoid body. 4. The process according to claim 3, wherein the differentiation of stem cells is carried out in an organoid body. 4. The process according to claim 3, wherein the stem cells secondary from the organoid body are cultured and differentiated. The method according to any one of claims 1 to 5, wherein a stimulation of the development of a pancreatic hormone producing pancreatic hormone is provided comprising stimulated proliferation of cells producing pancreatic hormone and / or stimulated differentiation of stem cells. Characterized by a process. The stimulus treatment of claim 6 wherein the stimulus is: Treatment with supernatant of the primary medium of the endocrine pancreas, Treatment with supernatants of the cell lines of the endocrine pancreas, Co-culture with differentiated cells of the endocrine pancreas, Coculture with cell lines of the endocrine pancreas, or Treatment with fixed molecular growth factors, Activation of at least one gene associated with differentiating stem cells into cells that produce pancreatic hormones, and Treatment with molecular growth factors dissolved in liquid And at least one of the following. 8. The process of claim 7, wherein treating with the immobilized molecular growth factor comprises cell imprinting with a molecular differentiation factor immobilized on a carrier. The process of claim 8, wherein a synthetic matrix, cell membrane or three-dimensional matrix substrate is used as the carrier. 10. The process according to any one of claims 1 to 9, wherein the identification and selection of cells producing pancreatic hormones is provided. The process of claim 10, wherein the selection of cells that produce pancreatic hormones comprises a cell sorting process. 12. The process of claim 10 or 11, wherein the cells that have not been identified and selected are further cultured and differentiated. The process according to any one of claims 1 to 12, wherein stem cells obtained from the glands of the organism or the glands of the gastrointestinal tract are used. The process according to claim 13, wherein stem cells obtained from the pancreas or salivary glands of the organism are used. The process according to any one of claims 1 to 14, characterized in that stem cells derived from glandular tissues, i.e., glandular tissues, are used. The process according to any one of claims 1 to 15, wherein stem cells derived from vertebrates, including mammals, are used. The process of claim 16, wherein stem cells derived from primates, including humans, are used. 18. The process according to any one of claims 1 to 17, wherein the cells producing the pancreatic hormone are used for pharmaceutical applications. 19. The process according to claim 18, wherein said pancreatic hormone producing cells are used for the treatment of pancreatic disease, metabolic syndrome or metabolic disease. 20. The process according to claim 19, wherein said pancreatic hormone producing cells are used for the treatment of diabetes mellitus, hyperglycemia or impaired glucose tolerance. 21. The process of any one of claims 1 to 20, wherein said pancreatic hormone producing cells produce insulin. An isolated pancreatic hormone-producing cell, characterized in that the cell is formed from stem cells isolated from differentiated exocrine glandular tissue of the organism. 23. The isolated pancreatic hormone producing cell of claim 22 wherein said isolated pancreatic hormone producing cell is a human cell. A cell composition comprising a plurality of cells producing the pancreatic hormone according to claim 22. The cell composition of claim 24, wherein the cells producing pancreatic hormone are formed by a process according to any one of claims 1 to 21. 26. The cell composition of claim 24 or 25, further comprising other cell types. 27. The cell composition of claim 26, wherein said other cell type comprises stem cells in pancreatic tissue and / or adjacent cells of Langerhans islet. 28. The cell composition of any one of claims 24 to 27 comprising an envelope or matrix material. Use of a cell according to claim 22 or 23 or a cell composition according to claim 24 as a pharmaceutical composition. Use of a cell according to claim 22 or 23 or a cell composition according to claim 24 as an autopharmaceutical composition. Use for implantation of a cell according to claim 22 or 23 or a cell composition according to claim 24 or in a medical device. An artificial Langerhans island comprising a cell according to claim 22 or 23 or a composition according to claim 24. A pharmaceutical composition comprising at least one cell according to claim 22 or 23 or at least one cell composition according to claim 24, and a pharmaceutical carrier substrate.

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