FR2722797A1 - CULTURE MEDIUM PROMOTING CELL GROWTH OF ENDOCRINE CELLS - Google Patents

Abstract

A culture medium essentially consisting of a combination of a basic nutritive mixture in which the amino acid valine is present in racemic form D, and a dialysed foetal calf serum complement, as well as at least one epidermal growth factor, cholera toxin and hormones. The culture medium may be used for culturing certain endocrine cells capable of incorporating and decarboxylating bioamine precursors. The resulting cells may be used as organ substitutes in transplants.

Description

 The present invention relates to a culture medium promoting cell growth of endocrine cells.

We know all the problems we encounter when we want to remedy organic, innate or acquired functional insufficiencies of the endocrine glands. Several solutions have been proposed which have proven insufficient for multiple reasons
- in certain cases, due to their instability, or the impossibility of producing them, the missing hormone (s) cannot be supplied in the form of a medicine
- when this or these hormones exist in the form of a drug, the chronology of their administration is delicate it is necessary to adapt it to the physiological variations of the regulated metabolisms
- transplantation of allogenic organs is impossible or at least highly improbable due to their very high immunogenicity
- normal endocrine cells, taken in isolation without any contamination by other cells of the tissue concerned, are non-immunogenic.

 The ideal solution would therefore consist in replacing the only cells concerned.

 To isolate these endocrine cells, it is necessary to dissociate the tissue concerned then prepare, by filtration or suitable centrifugation, a cell suspension as enriched as possible in endocrine cells.

 It is the cell culture of this suspension which constitutes the second essential step of this purification. This culture must be selective, in order to permanently conserve only the endocrine cells, it must be adapted to allow the conservation of the functional characteristics of these cells as well as their multiplication in vitro.

 At present, no culture medium fulfills these conditions.

The object of the present invention is to solve this problem by proposing a new culture medium consisting of the association of nutritional and stimulating elements capable of meeting the double requirement defined below.
To define this mixture, the inventors chose to consider the cells according to different approaches hitherto always dissociated
- the cells were considered according to their exodermal origin, which gives them requirements for epithelial cells
- they have been considered according to their belonging to the APUD system (Amine Precursor Uptake Decarboxylation or cells capable of incorporating and decarboxylating the precursors of biogenic amines).

These cells, like the cells of the mammary gland, are then able to use an amino acid substrate of racemic form D (such as D
Valine) ability not possessed by the most unwanted cells fibroblasts, macrophages
- They have been considered according to their endocrine nature, which leads to an enrichment of the nutritional mixture with several stimulating factors known for their influence on the expression of the functionalities of the differentiated cells.

 During their research, the inventors were able to determine that it was essential that this culture medium does not contain any trace of L Valine; they therefore, on the one hand replaced in the basic solution the L Valine by the D Valine and on the other hand used, for the complementation, fetal calf serum dialyzed, supplemented with growth factors and hormones.

The culture medium promoting cell growth of endocrine cells according to the invention therefore essentially consists of the combination of a basic nutritional mixture in which the amino acid Valine is provided in the racemic form D and a supplement thereof. dialyzed fetal calf serum, growth factors and hormones
This culture medium thus has the three desired properties
- selectivity
- response to the need for differentiating factors
- response to the need for stimulating factors
This medium is therefore especially suitable for the cell culture of certain endocrine cells.

According to a particular embodiment of the invention, the culture medium has the following composition
- MEM base solution (minimum essential medium) D Valine (Earle salt + D
Valine)
- complementation:
- Calysed calf serum (6 p.m. against a cellulose membrane
regenerated, porosity 1000 da) 0.02 to 0.20 ml / ml
- Epidermal growth factor 1 to 50 ng / ml - Insulin 0.001 to 20 sg / ml
- Hydrocortisone 0.004 to 5 Cig / ml
- Transferrin 0.5 to 100 Icg / ml
- Cholera toxin 10-8 to 10.12 M
- 3.3'-5 Triiodo L Thyronine 2.10 -7 to 2.10-11 M
- Bovine pituitary extract 0.05 to 100 ug / ml
This medium will be referred to hereinafter as APUDCM
According to another preferred embodiment of the invention, the culture medium has the following composition
- MEM D Valine base solution (Earle salt + D Valine)
- complementation:
- Calysed calf serum (1 8h against a regenerated cellulose membrane, porosity 1000 da) 0.12 ml / ml
- Epidermal growth factor 10 ng / ml
- Insulin 5 Eng / ml
- Hydrocortisone 400 ng / ml
- Transferrin 5 sg / ml
- Cholera toxin 10-10 M
- 3.3'-5 Triiodo L Thyronine 2.10 -9 M
- Bovine pituitary extract 5 stg / ml
This culture medium will be referred to hereinafter as PTCM.

 This medium has been used more particularly for the culture of parathyroid cells obtained from glands of hyperplastic adult subjects, adenomatous or normal, by enzymatic digestion, filtration or centrifugation in discontinuous Percoll gradient.

The present invention will be clearly understood with the aid of the following description which illustrates it without in any way limiting it, as well as the appended schematic drawing representing some of the results obtained. In the schematic drawing
Figure 1 shows the cell count curves
Figure 2 is a bar graph illustrating the dosage in
chemiluminescence for a primary culture of parathormone (PTH) in
culture supernatants
Figure 3 is a bar graph similar to bar graph 2 after
First passage
Figure 4 represents curves of modifications of the secretion of
PTH Induced by the variation of the extra cellular calcium level.

Obtaining cell preparation
Glandular samples were obtained, after consent of the patients concerned. After anatomo-pathological verification of the surgical piece in extemporaneous. the tissue fragment is placed in a sterile bottle containing physiological saline and then transported to the cell culture laboratory. It is then immediately treated or cryopreserved with a view to subsequent cultivation.

 After a fine cutting of the sample, the fragments obtained are rinsed to remove the residual blood and then weighed. One of these samples is passed to ultrasound to determine its parathyroid hormone content.

Tissue digestion is obtained by incubation 45 to 60 minutes at 37 "C, with mechanical agitation, by pipetting every 15 minutes in the following buffer
- 20 mM HEPES, 136 mM NaCI. 4.7 mM KCI. 0.65 mM MgSO4, 1.2 mM CaC12
- 1.5 mg / ml collagenase
- 40 stg / ml DNAse
- pH 7.45
The cell suspension obtained is filtered at 25 rpm and then incubated for 3 minutes in the following EGTA buffer.
-1 mM EGTA
- 20 mM HEPES, 142 mM NaCI, 07 mM KCI
- pH 7.4
This dissociates the cellular aggregates.

 The purification of the parathyroldian cells is carried out by centrifugation of the cell suspension on a discontinuous Percoll gradient. Diluting a Percoll isotonic stock solution (d = 1.123) with a Hanks saline solution (d = 1.006) makes it possible to obtain 3 solutions of density 1.035, 1.058 and 1.090. Centrifugation is carried out at 2000 rpm for 20 min with minimum acceleration and deceleration.

 The cells are gently collected at each interface, washed with all traces of Percoll and then counted. Cell viability is determined by exclusion with Trypan Blue. An aliquot of each suspension is kept to allow a measurement of its cellular PTH content.

Cellular culture
The cells are seeded at a density of 105 cells / cm 2 in 24-well plates and incubated for 18 hours at 37 ° C. in a base medium supplemented with 12? O of fetal calf serum. After this period, the medium is replaced by the PCTM environment.

 Every three days, the culture medium is removed, filtered, stored at -20 ° C. for the determination of PTH and replaced by fresh medium.

At confluence, the cells are detached by the action of a solution of trypsin-EDTA. Cell culture is then pursued in two forms
- seeding in 24-well plates at the rate of 1.5 × 10 5 cellsl well in order to estimate cell growth, the count is made 245 hours after seeding and then every 3 days in triplicate.

- seeding in Petri dishes. at the density of 105 cells / cm2 in order to continue the culture
Cell characterization The various cell characterization examinations were carried out according to the following techniques
- Light microscopy cell staining with the May Grunwald reagent
Giemsa allows to characterize the different types of parenchymal cells
- Electron microscopy, ultra-fine cell sections, observed in secondary culture, were observed
- Immunolabels:
Antikeratin characterization of the epithelial nature of cells
(mouse monoclonal antibody)
Anti Factor VIII Indication of the presence or absence of cells
endothelial cells in the cell monolayer (mouse monoclonal antibody)
Anti intact PTH highlighting of the endocrine character
parathyroid of cells in culture by intra-cytoplasmic labeling
(rabbit polyclonal antibody)
Functionality of cells in culture.

It has been studied as follows
- The assays of intact PTH were carried out by chemiluminescence at
using the "Magic Lite Intact PTH" dosing kit marketed by Ciba
Corning, both on the initial tissue and on the purified cells and in the
culture supernatants
- For the determination of the sensitivity to calcium, the cells, during the
primary culture and during the first transplant, were exposed for 2 hours
at variations in the concentration of extracellular calcium 'passing from 0.62
mM (base level in PTCM medium) at 1.8 mM. At the end of this incubation,
the supernatants were removed and the parathyroid hormone assay was performed
Immunoqenicity
- The expression of HLA DR antibodies has been studied by immunostaining
fluorescence using a mouse monoclonal antibody. This marking has
was carried out during all the stages of cell preparation, of the primary culture, during the first subculturing and after incubation for 48 hours of cell monolayers with 200 U / ml of interferon y.

- Mixed cultures cultures carried out with parathyroid cells irradiated at 2500 rad. according to the Saxony protocol (Surgery 1990 108: 56-62: "In vitro assessment of parathyroid Immunogenicity")
RESULTS.

Purification and cell culture
Percoll gradient centrifugation allowed the purification of endocrine parathyroldian cells.

 Aliquots of cell suspension, obtained after Percoll gradient centrifugation, were cytocentrifuged and fixed.

Staining with May Grunwald Giemsa reagent allowed the characterization of the isolated cell types for each density (x 100):
Phase contrast optical microscopy highlights the cell types conventionally described in parathyroid tissue.

- 1035 g / ml - mainly fat cells and cellular debris
-1.058 g / ml - small main cells with some oxyphilic cells
-1.090 g / ml - main cells.

 The viability of the fresh tissue is of the order of 80 to 90%; it decreases slightly for the cryopreserved tissue.

 30 to 40 C.c of the initial glandular material is recovered after centrifugation on Percoil 52.82 ng to 120.68 ng of PTH / g of initial tissue for 12.76 ng to 49.92 ng of PTH / g of cells after Percoll.

 Cell adhesion is obtained 24 hours after sowing in the PTCM medium with an adhesion capacity of the order of 70%. Clones typical of epithelial cells appear from the second day and a cell monolayer is obtained within 6 to 8 days. The few fibroblastic type cells visible in the first days (<1%) do not proliferate thereafter.

The cell count curves represented in FIG. 1 of the appended schematic drawing (the duration, in days, being plotted on the abscissa and the number of cells seeded on the ordinate) show that the number of cells seeded in primary culture is restored under 8-10 days. On curve A, the cell count was carried out in primary culture and on curve B in secondary culture at the first subculture. The results represent the average achieved for triplicate tests for 3 strains
independent cell phones.

On curve A, we see that on day D + 1 of the primary culture, 60 to 80% of the seeded cells have adhered. Their initial number is restored within 8 to 10 days
On curve B, it can be seen that the adhesion of the cells is reduced during the transition to secondary culture, on day D + 1, 30 to 50% of the cells adhere, however, on D + 10, we reach 80% of the initial number of cells.

The best results are obtained when the passage is made at subconfluence it was then possible to carry out 3 to 5 passages before the appearance of cellular senescence
Cell characterization
Examination by electron microscopy has demonstrated the endocrine nature of cells in culture. The ultra structure corresponds to that of main active cells containing secretory grains, a Golgi apparatus and an active endoplasmic reticulum, cells described in normal or parathyroid tissue. adenomatous. (Weymouth RJ and Sheridans MN - 1966 - Acta
Endocrinol. 53 529-546 and Thiele J Damer J. and Fischer R - 1988: - J.

Submicros. Cytol. Pathol. 20 (3) 491-500)
Immunostaining. carried out on primary cultures and during the first pass gave the following results
- In the case of human anti-keratin immunolabelling, there is a positive labeling of the intermediate filaments throughout the cell population the control carried out with a non-immune mouse serum in place of the first antibody is negative, which reveals the exclusively epithelial nature cells (Miettinen M. et al. 1985 - Arch. Pathol. Lab. Med. 109: 986-989).

 - In the case of anti-human factor VIII immunostaining, the absence of endothelial cells is observed during the cell culture phase. The specificity of the labeling is confirmed by the presence of positively labeled cells after the enzymatic digestion.

- In the case of intact human anti-PTH immunolabelling,
The anti-PTH antibody positively labels all the cells fixed both in primary culture and during the first pass, the controls carried out with rabbit serum in place of the first antibody do not reveal any fluorescence.

 The endocrine nature of the cells is thus demonstrated: each cell contains PTH, whether in primary culture or at the end of the first subculture, which expresses the maintenance of cell differentiation.

Functionality - The dosages of intact PTH are represented on the bar graphs of Figures 2 and 3 (the duration, in days, is plotted on the x-axis and and the secretion of PTH on the y-axis). In the two figures, A, B and C are 3 representative cell strains the results express the average of the tests carried out in triplicate for each gland. The determination of PTH in the culture supernatants was carried out by chemiluminescence
For FIG. 2, corresponding to the measurement of the secretion activity for 3 days of a primary culture, an average base rate of 34.66 pg / 105 cells / h is obtained for an extraceliular calcium concentration of 0, 62 mM.

For FIG. 3, after a first pass, there is an average secretion rate of 34.39 pg / 105 cells / h
The study of the modification of the secretion of PTH induced by the variation of the level of extracellular calcium is represented on the curves of FIG. 4 (the duration, in hours, is plotted on the abscissa and the secretion of PTH on the ordinate). On these curves the results represent an average of 3 independent tests, carried out in triplicate. The cells at confluence during the first passage were incubated for 2 h in PCTM medium at calcium levels of 0.62 mM (base rate) and 1.8 mM.

 We see that an increase in the calcium level from 0.62 mM to 1.8 mM induces a decrease in the secretion of intact PTH by about 40% of the base level, this modification corresponds to the normal regulation of parathyroldian cells in physiological conditions in vivo. (Brown et al. Am.

 J. Med. 66 923-931).

 Immunoqneicity - Some cells were positively labeled during anti-HLA DR immunolabelling performed at the various stages of cell purification.

 This positive labeling is no longer detected within the cell monolayer throughout the culture even after stimulation of the expression of these class II molecules by the interferon y.

The results presented in the following table show the capacity of the T lymphocytes to react to stimulation by Concanavalin A as well as their absence of reaction to the purified parathyroldian cells, which confirms the nonimmunogenicity of the latter.
Co-culture of sarathvroid cells (PT) -lvmphocvtes (Lvm)
Cell suspension Cells per well Incorporation of tritiated thymidine
Lym PT (pulses / min)
SEM average
PT irradiated 105 342 38
Lym / PT irradiated 105 105 444 175
Lym 105 148 94
Lym + Concanavalin A) i 105 1 1992 582
(0.25 mg / well)
The above description clearly shows the originality of the formulation of the proposed culture medium, which combines constituents known for their ability to maintain the properties of endocrine cells and to allow the proliferation of epithelial cells, with a particular amino acid, D
Valine which ensures, by eliminating parasitic fibroblast cells, macrophages, the selection of only endocrine cells.

 This original formulation allows the survival and cellular multiplication of purified, functional and non-immunocompetent parathyroid endocrine cells.

 The similarity of enzymatic equipment which allows the parathyroldian cells and the cells of the mammary gland to convert the amino acid D Valine and L Valine, as well as preliminary results with the t cells of the islets of Langerhaus suggests that this enzymatic equipment may be common to many endocrine cells.

 The cells obtained by culture on the medium according to the invention are capable of application by grafting as an organ substitute.

Claims (5)

 1 - Culture medium promoting cell growth of endocrine cells characterized in that it essentially consists of the association of a basic nutritional mixture in which the amino acid Valine is provided in the racemic form D and of a supplement in dialyzed fetal calf serum, growth factors and hormones.  2 - culture medium according to claim 1 characterized in that it has the following composition  - MEM D Valine base solution (Earle salt + D Valine)  - supplementation: - Calf serum dialyzed (18h against a regenerated cellulose membrane, porosity 1000 da) 0.02 to 0.20 ml / ml  - Epidermal growth factor 1 to 50 ng / ml  - Insulin 0.001 to 20 g / ml  - Hydrocortisone 0.004 to 5 tzg / ml  - Transferrin 0.5 to 100 ig / ml  - Cholera toxin 10-8 to 10-12 M  - 3.3'-5 Triiodo L Thyronine 2.10 ~ 7 to 2.10-11 M  - Bovine pituitary extract 0.05 to 100 ug / ml  3 - culture medium according to claim 2 characterized in that it has the following composition  - MEM D Valine base solution (Earle salt + D Valine)  - supplementation: - Calysed calf serum (18h against a regenerated cellulose membrane, porosity 1000 da) 0.12 ml / ml  - Epidermal growth factor 10 ng / ml  - Insulin 5 stg / ml  - Hydrocortisone 400 ng / ml  - Transferrin 5 ltg / ml  - Cholera toxin 10-1 M  - 3.3'-5 Triiodo L Thyronine 2.10 -9 M  - Bovine pituitary extract 5 tzg / ml  4 - Application of the culture medium according to any one of claims 1 to 3 to the cell culture of certain endocrine cells capable of incorporating and decarboxylating the precursors of biogenic amines  5. Cells obtained by culture on the medium according to any one of claims 1 to 4 for the preparation of organ substitutes.