WO1996003496A1

WO1996003496A1 - Culture medium promoting endocrine cell growth

Info

Publication number: WO1996003496A1
Application number: PCT/FR1995/000961
Authority: WO
Inventors: Marie Thérèse ZABOT; Jean Michel Zabot
Original assignee: De Leobardy, Francis; DUGAST, Hervé
Priority date: 1994-07-22
Filing date: 1995-07-18
Publication date: 1996-02-08
Also published as: FR2722797A1; AU3079995A

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

CULTURE MEDIUM PROMOTING CELL GROWTH

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 proved 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 present invention is given for the purpose of solving the problem before it by providing a new culture medium consisting of ^the association of nutrients and stimulants able to meet the dual requirements defined above.

To define this mixture, the inventors have chosen 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 capable of using an amino acid substrate of racemic form D (such as D Valine) capacity which the most undesirable cells do not have: fibroblasts, macrophages - do 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 L Valine by D Valine and on the other hand used, for the complementation, fetal calf serum dialysis, supplemented with growth factors and hormones.

Certain media have been proposed for the culture of mammary cells. Several culture media use MEM D Valine (CELL BIOLOGY INTERNATIONAL REPORTS, vol. 12 no.5 May 1988, pp. 355-364), optionally supplemented with fetal calf serum and insulin (IN VITRO Vol 14, no. 3, March 1978, Rockville MD US pp 271-281), (IN VITRO Vol 18, n ° 4, April 1982, Rockville MD US pp 407-414). US-A-4,423,145 describes conventional culture media, based on L Valine, associated with growth factors and hormones.

The culture medium promoting cell growth of endocrine cells according to the invention 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 in serum calf dialysis, 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 specially adapted to the cell culture of certain endocrine cells.

According to a particular embodiment of the invention, the culture medium has the following composition:

- basic solution: MEM (minimum essential medium) D Valine (Earle salt + D Valine)

- complementation:

- Calf dialysis 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 mg / ml

- Hydrocortisone 0.004 to 5 mg / ml - Transferrin 0.5 to 100 mg / ml

- Cholera toxin 10 -8 to 10 -12 M

- 3.3'-5 Triiodo L Thyronine 2.10 -i to 2.10 -11 M

- Bovine pituitary extract 0.05 to 100 mg / ml This medium will be called hereinafter: APUDCM According to another preferred embodiment of the invention, the culture medium has the following composition:

- basic solution: MEM D Valine (Earle salt + D Valine)

- complementation:

- Calf serum dialysis (18h against a regenerated cellulose membrane, porosity 1000 da) 0.12 ml / ml

- Epidermal growth factor 10 ng / ml

- Insulin 5 mg / ml

- Hydrocortisone 400 ng / ml - Transferrin 5 mg / ml - Cholera toxin 10 -ιo M

- 3.3'-5 Triiodo L Thyronine 2.10 -9 M

- Bovine pituitary extract 5 mg / ml

This culture medium will be called subsequently: PTCM.

This medium was 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 chemiluminescence assay for a primary culture of parathormone (PTH) in culture supernatants

Figure 3 is a bar graph similar to bar graph 2 after first pass

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 affected patients. After anatomo-pathological verification of the surgical patch in extemporaneous, the tissue fragment is placed in a sterile bottle containing physiological serum and then transported to the cell culture laboratory. It is then immediately treated or cryopreserved for 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 in order to determine its parathormone content.

Tissue digestion is obtained by incubation 45 to 60 minutes at 37 ° with mechanical agitation, by pipetting every 15 minutes in the following tampon - 20 mM HEPES. 136 mM NaCI, 4.7 mM KCI, 0.65 mM MgS04, 1 , 2 m

CaCI2

- 1.5 mg / ml collagenase

- 40 mg / ml DNAse

- pH 7.45 The cell suspension obtained is filtered at 25 mm 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 The cell aggregates are thus dissociated.

The purification of the parathyroid 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 Bleu Trypan. An aliquot of each suspension is kept to allow a measurement of its cellular PTH content. Cell culture The cells are seeded at a density of 105 cells / cm2 in 24-well plates and incubated for 18 hours at 37 ° C in a base medium supplemented with 12% fetal calf serum. After this period, the medium is replaced by the PCTM medium.

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

- inoculation in 24-well plates at a rate of 1, 5.105 cells / well in order to estimate cell growth; we count 245 hours after

_! £> seeding then every 3 days in triplicate.

- seeding in Petri dishes, at a 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: ₁₅ - Optical 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:

₂₀ Antikeratin: characterization of the epithelial nature of cells (mouse monoclonal antibody)

Anti Factor VIII: indication of the presence or absence of endothelial cells in the cell monolayer (mouse monoclonal antibody)

Intact anti-PTH: demonstration of the endocrine parathyroid character 2 & of the cells in culture by intra-cytoplasmic labeling (rabbit polyclonal antibody)

Functionality of cells in culture. It has been studied as follows:

- assays intact PTH was made in chemiluminescence ₀ to 3 using the assay kit "Magic Lite Intact PTH" sold 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 subculture, were exposed for 2 hours to 35 variations in the concentration of extracellular calcium: passage of 0.62 mM (base rate in PTCM medium) at 1.8 mM. At the end of this incubation, the supernatants were removed and the assay of the parathormone

Immunoqenicity

- The expression of H LA DR antibodies was studied by fluorescent immunostaining using a mouse monoclonal antibody. This labeling 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 the cell monolayers with 200 U / ml of interferon g.

- 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 parathyroid 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% of the initial glandular material is recovered after centrifugation on Percoll: 52.82 ng to 120.68 ng of PTH / g of initial tissue for 12.76 πg 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 carried out for triplicate tests for 3 independent cell strains. , 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, at D + 10, 80% of the initial number of cells is reached.

The best results are obtained when the transition is made to subconfluence; it was then possible to make 3 to 5 passages before the appearance of cellular senescence.

Cell characterization: Examination by electron microscopy demonstrated the endocrine nature of cells in culture. The ultra structure corresponds to that of active main cells containing secretory grains, a Golgi apparatus and an active endoplasmic reticulum, cells described in normal or adenomatous parathyroid tissue. (Weymouth R.J. and Sheridans M.N. - 1966 - Acta Endocrinol. 53: 529-546 and Thiele J. Dârner J. and Fischer R - 1988: - J. Submicros. Cytol. Pathol. 20 (3): 491-500).

Immunostaining, performed 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 of the 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 immunostaining, the anti-PTH antibody positively labels all the cells fixed both in primary culture and during the first passage; the controls carried out with a rabbit serum in place of the first antibody do not reveal any fluorescence. We thus highlight the endocrine nature of cells: 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 graph 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 assay 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 is obtained, 6 pg / 105 cells / h for an extracellular calcium concentration of 0.62 mM.

For FIG. 3, after a first pass, an average secretion rate of 34.39 pg / 105 cells / h is noted. The study of the modification of the secretion of PTH induced by the variation of extracellular calcium level 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 level and 1.8 mM.

It can be seen that an increase in the level of calcium from 0.62 mM to 1.8 m induces a decrease in the secretion of intact PTH by approximately 40% of the basic rate. This modification corresponds to the normal regulation of the parathyroid cells in the physiological conditions in vivo. (Brown et al. Am. J

Med. 66: 923-931).

Immunogenicity - Some cells were positively labeled during anti-HLA DR immunostaining 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 this class II molecules by interferon g.

The results presented in the following table show the capacity of T lymphocytes to react to stimulation by Concanavalin A as well as their absence of reaction to purified parathyroid cells, which confirms the no immunogenicity of the latter. Co-culture of parathyroid cells (PTHvmphocvtes (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) 105 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 cells: fibroblasts, 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 parathyroid 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 b 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

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 fetal calf serum dialysis, supplemented with at least one epidermal growth factor, cholera toxin and hormones.

2 - Culture medium according to claim 1 characterized in that it has the following composition:

- basic solution: MEM D Valine (Earle salt + D Valine) - supplementation: - Calf serum dialysis (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 mg / ml

- Hydrocortisone 0.004 to 5 mg / ml - Transferrin 0.5 to 100 mg / 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 mg / ml

3 - Culture medium according to claim 2 characterized in that it has the following composition:

- basic solution: MEM D Valine (Earle salt + D Valine) - supplementation: - Calf serum dialysis (18h against a regenerated cellulose membrane, porosity 1000 da) 0.12 ml / ml

- Epidermal growth factor 10 ng / ml - Insulin 5 mg / ml

- Hydrocortisone 400 ng / ml

- Transferrin 5 mg / ml

- Cholera toxin 10 -10 M

- 3.3'-5 Triiodo L Thyronine 2.10 -9 M - Bovine pituitary extract 5 mg / 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 use as an organ substitute.