JPS5871885A - Established human somatotropin-producing cell strain - Google Patents

Abstract

PURPOSE:To provide established human somatotropin-producing cell strain originated from the human pituitary gland tissue and capable of producing human somatotropin stably for a long period. CONSTITUTION:Human pituitary gland tissue extirpated from the human body, e.g. pituitary adenoma tissue extirpated from the patient of acromegaly by operation, is dispersed in a proteinase solution, e.g. a solution containing 50-1,200 protease units of dispase per ml. The dispersed cells are added to a culturing medium containing F-10 medium and 1-25vol% serum, preferably a mixed serum of bovine neonate serum, bovine fetus serum and equine serum. The proliferated epithelial cells are transplanted in the body of an animal having lowered immunity, e.g. nude mouse, nude rat, etc. After proliferating the transplanted cells in the body, the cells are extirpated from the body, and dispersed and cultured again in the above proteinase solution and culturing medium.

Description

DETAILED DESCRIPTION OF THE INVENTION 10. Background Technical Field of the Invention The present invention relates to an established human growth hormone producing cell line.

More specifically, the present invention involves culturing human pituitary tissue extracted from a human body in a specific medium, transplanting the obtained epithelial cell group into an animal body with reduced immune capacity, and culturing the transplanted cells grown in the animal body. The present invention relates to a newly established human growth hormone-producing cell line obtained by excising the cell line and culturing it again in a specific medium.

Prior Art Conventionally, human growth hormone has been obtained by extracting the pituitary gland removed from a human cadaver with a solvent. However, due to the limited amount of pituitary gland that can be obtained using this method, it has not been possible to supply a sufficient amount of human growth hormone, which is medically necessary.

Therefore, various methods for obtaining large amounts of human growth hormone have been studied, and methods using genetic engineering techniques are being developed, but so far, they have not been put into practical use due to low yields and high costs. In addition, many attempts have been made to obtain human growth hormone by culturing human pituitary cells, but in all cases, the ability to secrete the hormone is lost early in the culture.
There have been no reports of success in obtaining an established cell line that stably produces the hormone.

(9) Purpose of the Invention The purpose of the present invention is to provide an established human growth hormone cell line that stably produces human growth hormone over a long period of time.

(1) Detailed Description of the Invention The present invention consists of human growth hormone cell lines described in the following sections.

(1) An established human growth hormone producing cell line derived from human pituitary tissue.

(2) Dispersing human pituitary tissue extracted from the human body with a protease solution, adding the obtained dispersed cells to a culture medium containing serum and Ham's F-10 medium, and culturing the cells,
The proliferated epithelial cell group is transplanted into an animal body with reduced immunocompetence, the transplanted cells proliferated in the animal body are excised, and the excised transplanted cells are redispersed using a protein hydrolytic enzyme solution and the above culture medium. The above-mentioned first strain was established by culturing
The human growth hormone-producing cell line described in Section 2.

(3) The human growth hormone producing cell line according to item 2 above, wherein the ratio of serum in the culture solution is 1' to 25 cents (volume).

(4) The human growth hormone producing cell line according to item 2 or 3 above, wherein the serum is a mixed serum of newborn bovine serum, fetal bovine serum and horse serum.

(5) The human growth hormone producing cell line according to item 4 above, wherein the mixing ratio (volume) of newborn bovine serum, fetal bovine serum and horse serum is 4:2:1.

(6) The human growth hormone producing cell line according to item 2 above, wherein the culture medium contains an antibiotic.

(7) The human growth hormone producing cell line according to item 6 above, wherein the culture solution has the following composition.

Ham F-10825 parts (volume) Newborn bovine serum 100〃 Fetal bovine serum 50〃 Horse serum 25〃 Penicillin 50 units/-Streptomycin 50 μP/mJ.

(8) Is the protease solution containing 50 to 1200 protease units/-? - the above-mentioned second solution which is
The human growth hormone-producing cell line described in Section 2.

(9) The human growth hormone-producing cell line according to item 8 above, wherein the protease solution is a nispase solution containing 3007' rotiase units/-.

(IQ, the human growth hormone-producing cell line according to item 2 above, wherein the immunocompromised animal is a nude mouse or nude rat.

The human growth hormone producing cell line according to item 2 above, wherein the culture of the excised transplanted cells is immunologically selective culture using lung antibodies and complement from the transplanted animal.

The human growth hormone producing cell line according to any one of the above items 1 to 11, wherein the human pituitary tissue is a pituitary adenoma tissue removed during surgery from a patient with acromegaly.

α field The human growth hormone producing cell line according to any one of the above items 1 to 12, having the following characteristics.

a) It is subcultured in a culture medium containing serum and Ham's F-10 medium; b) It has an epithelial-like morphology, but has weak adhesive properties and becomes floating before forming a cell sheet. cells appear; C) the distribution of chromosome number is hypoploid with a mode of 39; d) multicellular growth is promoted by adding insulin to the above culture solution; e) in the above culture solution culture does not produce human growth hormone, and stimulants such as insulin, hydrocortiscin, and human hypothalamic median eminence extract (rnedia emis) are used as stimulants.
When ence extract (MEE) or calcium ionophore is added to the culture solution, human growth hormone is produced;
Releases nt/ld human growth hormone.

To obtain the human growth hormone producing cells of the present invention, first,
Human pituitary tissue removed from the human body is cut into pieces as necessary and placed in a protease solution, and pituitary tissue cells are dispersed in the solution.

As the human pituitary gland tissue, those excised during surgery from patients with pituitary adenomas such as gigantism or acromegaly, those excised from human fetuses resulting from miscarriage, or from human cadavers, which have bioactivity, are used.

Examples of proteases used as cell dispersants include dis-4-ase and the like. The concentration of the enzyme solution is
50 to 1200 zolotease units/-, preferably about 300 grotease units/-.

The thus obtained dispersed cells were mixed with serum and Nomu F-1.
The cells are cultured in a culture solution containing 0 medium.

In addition, the media include Normu F-12, DM160, and R.
PMI 1640 etc. can also be used, but No.1 F-
10 is most preferred.

The culture medium used in this step is serum and Nomu F-10.
This is a new medium that has not been described in any literature.

The ratio of serum in the culture solution is not particularly limited, but is preferably 1 to 25% (by volume). As the serum, newborn bovine serum, fetal bovine serum, horse serum, etc. are used, and a mixed serum of these is particularly desirable. The mixing ratio is preferably neonatal bovine serum: fetal bovine serum: horse serum = 4:2:1.

The culture medium preferably contains serum and Ham's F-10 medium, as well as antibiotics such as penicillin and streptomycin.

The composition of Ham's F-10 medium according to the present invention is as follows.

Ham F-10 medium composition (rv/1000-) NaC17
400 KC1285 CaC42-2H2044 Ngso4-7H2o 153Na2H
PO4-7H20290 KH2PO483 NaI(COs 1200F e
S O40-834 Cu S O40-0025 znSO40,0288 Glucose 1100 L-Alanine 89 L-Arginine Hydrochloride 211 L-Athnogragin H2015, OL-Athnogragin Acid 13,3L-Cystine Hydrochloride
31.5L-glutamic acid 14.7L
-Glutamine 146.2 Glycine
7.5 L-Histidine 21. OL-inleucine 2.6L-leucine
13. IL-lysine hydrochloric acid 29.3L-
Methionine 4.48L-Phenylalanine 4.96L-Proline
11.5L-serine 10.5L-threonine 3.57L-tryptophan 0.60L-tyrosine
1.81L-valine 3.5 biotin 0.024/? Calcium Ntothenate 0.715 Choline Hydrochloride
0.698 Folic acid 1.32 Hypoxanthine 4.08 Myo-inositol 0.541 Niacinamide
0.615 Pyridoxine Hydrochloride 0
．． 206 Rideflavin 0.376 Thiamine Hydrochloride 1.012 Thymidine
0.727 Vitamin B, 21.36 Lipoic acid 0.2 Sorg pyruvate 110 Phenol Red 1.2 The culture in this step is carried out in the same manner as normal tissue culture except that the above culture solution is used. That is, under aerobic conditions, the pH is approximately 7.0.
~7.5, preferably pH 7.3-7.4, temperature 37″C
, to culture. It is most desirable to culture in an environment with 95-97% air and 3-5% CO2.

In this culture process, epithelial cells and fibroblasts grow in layers, so epithelial cells, known as hormone-producing cells, are separated and transplanted into the body of an animal with reduced immune capacity. , grown within the animal body. Even if fibroblasts are contaminated during transplantation, 1. Since these cells do not proliferate and die within the body of the transplanted animal, they do not pose any particular problem. Examples of immunocompromised animals include nude mice or nude rats. Transplantation is usually done subcutaneously in the back or intraperitoneally. The expanded cells can also be transplanted and passaged into new animals. The proliferating cells thus obtained are excised, redispersed and cultured using a proteolytic enzyme solution and the above-mentioned culture medium to obtain established human pituitary tissue-derived cells.

If the established cells are contaminated with cells derived from the transplanted animal, those cells are removed by immunological selection culture using the lung antibody and complement of the transplanted animal. For example, adding anti-spleen serum and complement prepared using rabbits to the culture medium during cell culture,
The cells are cultured for several hours, and then subcultured according to a conventional method. Cells from the transplanted animal are destroyed by antibodies and complement in the antiserum, and only human-derived cells proliferate.

The established human growth hormone producing cell line thus obtained has the following characteristics.

a) It is subcultured in a culture medium containing serum and Ham's F-10 medium; b) It has an epithelial-like morphology, but has weak adhesive properties and becomes floating before forming a cell sheet. Cells appear; C) As shown in Figure 1, the chromosome distribution is hypoploid with 39 modes; d) Add 0.01 to 100 μP/d of insulin to the culture medium. This promotes cell proliferation,
The effect becomes maximum at an addition amount of -10μ.

e) Human growth hormone was not produced when cultured in the above culture solution, and insulin (Figure 2), hydrocortiscin (Figure 3) and human hypothalamic median eminence extract (Figure 4) were used as stimulants. When added to the culture solution, human growth hormone is produced.

f) When implanted subcutaneously in a nude mouse, it forms a tumor and releases about 10-20 n%/mg of human growth hormone into the nude mouse serum.

g) Cryopreservation is performed in liquid nitrogen using a culture medium supplemented with 13-tidimethyl sulfoxide.

Human growth hormone can be produced by culturing the established human growth hormone-producing cell line obtained by culturing in the above-mentioned culture medium to which the above-mentioned stimulant has been added, and the hormone itself can be cultured by a known method. Collected from liquid.

Next, the present invention will be explained in more detail with reference to Examples.

Example: Pituitary adenoma tissue removed during surgery from a patient with acromegaly was immediately washed with Hank's salt solution supplemented with antibiotics.
Cut into small pieces using a razor blade. The thin sections were placed in a culture solution with the composition shown in Table 1 containing 300f rotiase units/-. -se (manufactured by Godo Shusei Co., Ltd.) and incubated at 37°C for 30 minutes, followed by light pipetting to disperse the cells. After centrifugation, the obtained cells were resuspended in the above culture medium, and 60%
Plant it in a petri dish (manufactured by Terumo Corporation) and perform primary culture. When the implanted cells begin to divide and proliferate and form a cell sheet containing a mixture of epithelial cells and fibroblasts, 0.
Using a piece of sterile paper impregnated with 25% trypsin solution, pick up only the epithelial cells and culture them separately. After subculture, nude mice (BALB/cA-nu/JCL
■Clair Japan Japan 2-7 The amount of human growth hormone secreted in the culture supernatant of pituitary adenoma-derived epithelial cells immediately before transplantation is 80 per ml in 24 hours.
It was 0 to 2400n%. One month after transplantation, a tumor is formed under the skin of the nude mouse, and a cell line that can be transplanted and passaged is obtained. At this time, the human growth hormone in the nude mouse serum was 1-10 to 20nf! -Measured. The tumor is removed, treated with enzymes in the same manner as the primary culture method described above, and returned to the culture system. This tumor contains not only human pituitary adenoma-derived cells but also nude mouse-derived cells such as vascular endothelial cells, blood cells, and stromal cells. Isolate adenoma-derived cells. Immunological selective culture is performed by selectively destroying nude mouse-derived cells using anti-nude mouse spleen antibodies obtained from domestic rabbits and complement. The separated cells were cloned and the JH8A-2 strain was established.

Although the JH8A-2 strain has an epithelial-like morphology, its adhesion is weak, and some cells appear in a floating state before forming a cell sheet. The doubling time of cell division is about 35 hours, and the addition of insulin to the culture medium has the effect of promoting proliferation. When adding insulin at a concentration of 0.01 μV- to 100 μV-, proliferation is observed at any concentration, with the greatest effect at 10 μf%/-. The chromosome number distribution is hypoploid with 39 modes, with approximately 901 cells distributed in the range of 34 to 41. Next, in terms of hormone secretion ability, this cell line does not secrete human growth hormone, with a culture supernatant of 1-9In7 or less after 20 hours of culture in the above-mentioned culture solution, and no intracellular content was measured. However, when a human hypothalamic median eminence extract is added to the culture solution, a remarkable secretion of human growth hormone of more than 50 On5L is observed under the same conditions. Such a human growth hormone secretion promoting effect on the JH8A-2 strain is caused not only by the human hypothalamic median eminence extract but also by insulin, hydrocortiscin, and calcium ionophore. For insulin, the above conditions (2
0 hour culture, culture supernatant 1 - about 300 nf, and about 200 nf for hydrocortiscin by adding 100 nM to the culture medium under the above conditions, respectively.
Approximately 50 of Ig-, and for calcium ionophore, 1 μ51'-/ld can be added to the culture medium.
Approximately 500 njiL of human growth hormone is measured per culture supernatant after hours of culture. Pituitary sex hormones other than human growth hormone, such as thyroid stimulating hormone, luteinizing hormone, follicle stimulating hormone, and adrenal hormone stimulating hormone;
Regarding prolactin, no secretion of any hormone was observed in the culture supernatant of 20-hour culture when 1 μm/- of human hypothalamic median eminence extract was added to the culture medium.

Table 1 Culture solution composition (in IA) Ham F-10825d □ Neonatal bovine serum 100- Fetal bovine serum 50- Horse serum 25d Penicillin 50 units/- Strenotomycin 50μ? Effects of the Invention According to the present invention, as described above, an established human growth hormone-producing cell line derived from human pituitary tissue is provided, and this cell line particularly has the following characteristics.

&) It is subcultured in a culture solution containing serum and Nomu F-10 medium; b) It has an epithelial-like morphology, but has weak adhesive properties and remains in a floating state before forming a cell sheet. C) The chromosome number distribution is hypoploid with a mode of 39; d) Cell proliferation is promoted by adding insulin to the above culture solution; e) In the above culture solution Human growth hormone is not produced when cultured in this culture, but human growth hormone is produced when insulin, hydrocortisone, human hypothalamic median eminence extract, or calcium ionophore is added to the culture solution as a stimulant.

f) When implanted subcutaneously in a nude mouse, it forms a tumor and releases about 10 to 20 np/d of human growth hormone into the nude mouse serum.

The human growth hormone producing cell line of the present invention maintains stable human growth hormone producing ability over a long period of time. Therefore, by culturing this cell line, Schicht's growth hormone can be obtained at low cost and in large quantities, and a sufficient amount can be supplied to patients who require the hormone. Furthermore, since the cell line of the present invention is a human-derived cell, there is no contamination of foreign proteins into human growth hormone preparations, it is easy to purify, and it can be safely administered to the human body. moreover,
Since the cell line of the present invention has a stable ability to produce human growth hormone under the skin of nude mice, it may be possible to transplant the cell line into the human body as an artificial pituitary gland and use it for treatment.

[Brief explanation of drawings]

Figure 1 shows the chromosome number distribution of the JH8A-2 strain, and Figures 2 to 4 show the presence of insulin, hydrocortiscin, or human hypothalamic median eminence extract in the culture medium of the JH8A-2 strain, respectively. The amount of human growth hormone (GH) produced when (MEE) is added is shown. Figure 1 U Misaki Color I book, 41ζ. Figure 2 rg/ml GH Insuiin ) ug/ml Figure 3 ng/ml GH H7drOCOrtlSOne nM Factor 4 ng/mt aH MEE ) ig/ml 401

Claims (1)

[Scope of Claims] (1) An established human growth hormone-producing cell line derived from human pituitary tissue. (2) Human pituitary tissue extracted from the human body is dispersed in a protease solution, and the resulting dispersed cells are mixed with serum and Ham's F-
The cells are cultured in addition to a culture solution containing 10 medium, and the proliferated epithelial cell group is transplanted into an animal body with reduced immune capacity,
Human growth according to claim 1, which is established by extracting the transplanted cells that have proliferated in the animal body, and redistributing and culturing the extracted transplanted cells using a protease solution and the above-mentioned culture solution. Hormone-producing cell line. (3) The ratio of serum in the culture solution is 1 to 25 cents (
3. The human growth hormone producing cell line according to claim 2, which has the following properties: (4) If the serum is neonatal bovine serum, fetal bovine serum or □
The human growth hormone producing cell line according to claim 2 or 3, which is a mixed serum of horse serum. (5) The human growth hormone producing cell line according to claim 4, wherein the mixing ratio (volume) of newborn bovine serum, fetal bovine serum and horse serum is 4:2:1. The human growth hormone producing cell line according to claim 2, which contains an antibiotic. (7) The human growth hormone producing cell line according to claim 6, wherein the culture solution has the following composition. Ham F-10825 parts (volume) Newborn bovine serum 100〃 Fetal bovine serum 50〃 Horse serum 25〃 (Nigirin 50 units/1n
l Stretutomycin 50 μl/- (8)
Claim 2, wherein the protease solution is a dispase solution containing 50 to 1200 protease units/-.
The human growth hormone-producing cell line described in Section 2. (9) The human growth hormone producing cell line according to claim 8, wherein the protease solution is a dispase solution containing 300 protease units/7. The human growth hormone producing cell line according to claim 2, wherein is a nude rat. (11) The human growth hormone producing cell line according to claim 2, wherein the culture of the excised transplanted cells is immunologically selective culture using pancreatic antibodies and complement from the transplanted animal. The human growth hormone producing cell line according to any one of claims 1 to 11, wherein the human pituitary tissue is a pituitary adenoma tissue removed during surgery from a patient with acromegaly. 03 The human growth hormone producing cell line according to any one of claims 1 to 12, which has the following characteristics. a) It is subcultured in a culture medium containing serum and Ham's F-10 medium; b) It has an epithelial-like morphology, but has weak adhesive properties and becomes floating before forming a cell sheet. cells appear; C) the chromosome number distribution is hypoploid with a mode of 39; d) cell proliferation is promoted by adding insulin to the above culture solution; e) in the above culture solution However, human growth hormone is produced when insulin, hydrocortiscin, human hypothalamic median eminence extract, or calcium ionophore is added to the culture medium as a stimulant.f) When transplanted subcutaneously into a nude mouse, it forms a tumor and releases about 10 to 20 nf/d of human growth hormone into the nude mouse serum.