JPH08140692A - Production of von wilebrand factor - Google Patents

Abstract

PURPOSE: To efficiently obtain the subject protein for treatment, etc., of von Wilebrand disease which is a congenital hemorrhagic disease in large amounts by culturing a vascular endothelial cell attached onto a microcarrier while adding shearing stress in a specific range. CONSTITUTION: This von Wilebrand factor useful for treatment, etc., of von Wilebrand disease which is a congenital hemorrhagic disease is efficiently obtained by culturing a vascular endothelial cell separated by from human umbilical vein an oxygen perfusion method in a cultured medium to which cultured supernatant of a human normal diploid fibroblast and fetal calf serum are added in a flask coated with collagen, and putting the culture into a spinner culturing bottle containing a microcarrier such as gelatin beads, inoculating a vascular endothelial cell thereinto, setting the number of rotation of stirring to 50-600rpm at 37 deg.C and culturing the cell for 8 days while applying shearing stress within a range of 20-130dyne/cm<2> . Thereby, mass production of the von Wilebrand factor by the cultured cell which has been difficult hitherto is made possible.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing von Willebrand factor, which may be used as a medicine, by using vascular endothelial cells.

[0002]

BACKGROUND OF THE INVENTION von Willebrand factor is one of plasma glycoproteins and is considered to be useful for the treatment of congenital bleeding disorder von Willebrand disease. Is known to be effective. However, since this preparation uses blood as a raw material, there is a possibility of contamination with risk factors such as hepatitis virus and AIDS virus, and there is concern about supply of raw materials. On the other hand, von Willebrand factor is a multimer in which a glycoprotein monomer having a molecular weight of 270 kDa is associated, and although the cDNA of the monomer has been cloned, expression of an active recombinant has not been achieved.

[0003]

DISCLOSURE OF THE INVENTION The present invention is directed to large-scale culture of vascular endothelial cells and efficient production of von Willebrand factor by vascular endothelial cells.

[0004]

It has been found that the above-mentioned object, that is, the large-scale culture of vascular endothelial cells and the efficient production of von Willebrand factor by vascular endothelial cells can be solved at once by the means described below. Was completed.

That is, the present invention relates to a method for culturing vascular endothelial cells previously devised by the present inventors (JP-A-4-17947).
By applying the method 6) to the production of von Willebrand factor, it has succeeded in solving the problem, and the vascular endothelial cells adhered on the microcarriers can be mixed in the range of ²⁰ to 130 dynes / cm ² by stirring the culture solution. The von Willebrand factor is produced by culturing while applying shear stress to produce the von Willebrand factor.

[0006] Vascular endothelial cells are cells that form the innermost layer of the inner wall of blood vessels, and control many substance-transmission between blood and tissues, and many physiologically active substances (colony stimulation) related to blood coagulation inhibition, hematopoiesis, blood pressure regulation and the like. Factors, interleukins, endothelin, plastaglandins, etc.) are produced and are deeply involved in the maintenance of the living body. In addition, proliferation of vascular endothelial cells is essential for repair of damaged blood vessels and angiogenesis in tissues.

In order to effectively utilize the functions and products of human vascular endothelial cells, it is necessary to culture and proliferate these cells in vitro. However, human vascular endothelial cells require growth factors and extracellular substances such as collagen. There is a matrix requirement, and it has been considered difficult from the economical aspect. Furthermore, vascular endothelial cells are adhesion-dependent, and monolayer culture conditions have generally been applied.However, under these conditions, their growth properties are weak even in the presence of growth factors and extracellular matrix, and substance production is weak. It was thought to be almost impossible to use for.

[0008] It has been confirmed that endothelial cells, which have been subjected to appropriate shear stress and cultured in microcarriers, survive and maintain for 7 days or more even under serum-free conditions after being grown to confluence, unlike the growth phase.

The production of von Willebrand factor is preferred because it is more enhanced in the serum-free condition than in the case of using a serum-containing culture medium under shear stress.

The points of success or failure of mass culture of cells are:
First, it must be able to grow 2. It is a point that it can be subcultured, and it is a long-term bone marrow cell culture system (so-called Dext
There are examples of blood stem cells by er cueture). Furthermore, subculture allows large-scale culture, but in many cases, the properties of the cells are changed, and, for example, the productivity of the target substance is reduced and the practical meaning is lost.

Under such circumstances, the present invention succeeds in subculturing human vascular endothelial cells, which have great practical significance, on a microcarrier containing an extracellular matrix component, and further, subculturing the cells. It was found that the repeated von Willebrand factor-producing ability of the cells also maintained a certain level, thereby providing a basis for the production of von Willebrand factor by cultured vascular endothelial cells.

The present invention will be specifically described below with reference to examples.

[0013]

【Example】

Example 1 Vascular endothelial cells separated from human umbilical vein by oxygen perfusion method were treated with fetal bovine serum 1 in a collagen-coated flask.
The cells were cultured and grown in M199 medium supplemented with 20% of the culture supernatant of human diploid fibroblasts containing 0% and ECGF activity. 200 ml of the above culture solution was placed in the spinner culture bottle (500 ml volume) shown in FIG. 1 containing gelatin beads (Gelibead, Hazleton) used as a 0.3 wt% microcarrier, and 1 endothelial cell grown in flat culture was added.
The cells were inoculated so that the concentration was × 10 ⁵ cells / ml. 37 ° C
The stirring speed was set to 50 to 600 rpm (11 to 128 dynes / cm ² ) and the cells were cultured for 8 days to examine their growth. The results are shown in FIG. 2. From this, the proliferation of vascular endothelial cells is
It was shown to depend on shear stress.

Example 2 Human umbilical vein-derived vascular endothelial cells were repeatedly exchanged with a medium at a frequency of once every 2 days in a spinner culture bottle under the shear stress condition of 300 rpm and 65 dynes / cm ^{2 in} the same manner as in Example 1. The cells were cultured for 8 days and grown to confluence.
The rotation was stopped, the gelatin beads to which the cells were attached were sedimented, the culture supernatant was removed, and the cells were washed 3 times with Dulbecco's phosphate buffered saline (PBS-).

Cells are released from gelatin beads using a cell detachment solution containing 0.25% trypsin. The released cells were counted, inoculated into a spinner culture bottle containing new gelatin beads at 1.5 × 10 ⁵ cells / ml, and the culture was continued again under the above-mentioned conditions, and this operation was repeated. As shown in FIG. 3, the cell arrival density was about 6.0 × 10 ⁵ cells / ml until the 12th passage.
The cell growth rate was about 4-fold every 8 days, but thereafter, both the cell growth rate and the cell arrival density decreased. The results of quantifying the amount of von Willebrand factor in the culture solution before each passage by enzyme immunoassay are shown in FIG. There was no significant change in the production of von Willebrand factor until at least the 9th passage.

This suggests that most of the cells cultured by at least the 9th passage operation are vascular endothelial cells and not fibroblasts which may possibly be contaminated. Furthermore, in the present example, since the same size spinner culture bottle was used for each passage, only 1/4 of the cells obtained in each passage were used, but all cultured cells were used without waste, Assuming that the scale of the culture is continuously expanded, the calculation will reach 1000 liters or more after 9 passages, which is a level that can be sufficiently considered as a substance production system.

Example 3 In the same manner as in Example 2, gelatin beads to which vascular endothelial cells adhered, which were obtained by culturing for 8 days, were precipitated and serum-free,
The cells were washed three times with M199 medium containing no human normal diploid fibroblast culture supernatant, and then again suspended in 200 ml of M199 medium containing no serum and no human normal diploid fibroblast culture supernatant. 6 ml of uniformly suspended suspension (6 ml each)
After adding 3 pieces of petri dish of 3 m and homogenizing the gelatin beads flatly, 37 ° C in a carbon dioxide gas incubator, 5
Static culture was performed for 5 days under the condition of% CO ₂ . The remaining suspension (about 180 ml) is 300 r in a spinner culture bottle.
Culturing was continued at 37 ° C. under the conditions of pm and 65 dynes / cm ² . On the 0th, 1st, 2nd and 5th day of culture, 0.2 ml each of the culture supernatant was collected from the dish and 1 ml was collected from the spinner culture bottle, and the amount of von Willebrand factor was measured by enzyme immunoassay for each. The results obtained are shown in FIG. As is clear from the figure, von Willebrand factor is almost not produced in 2 days under static culture conditions, whereas it is linearly produced until at least 5 days under shear conditions in spinner culture. Continuing, on the 5th day, it became more than 3 times as much as the static culture condition, and the concentration per culture solution was 14 μg / ml, which greatly increased the utility value as a raw material of the von Willebrand preparation.

[0018]

Industrial Applicability According to the present invention, it has become possible to mass-produce von Willebrand factor using cultured cells, which has been difficult in the past. The present invention is considered to be applicable to the culture of endothelial cells in which the gene of von Willebrand factor is recombined, and has a possibility of providing a more efficient method for producing von Willebrand factor.

[Brief description of drawings]

FIG. 1 shows a culture bottle preferably used in the present invention, in which a indicates the distance from the tip of the stirring bar to the inner wall of the bottle, and b indicates the inner diameter of the culture bottle.

FIG. 2 shows the effect of shear stress on the proliferation of vascular endothelial cells in Example 1.

FIG. 3 shows growth curves for subculture of human umbilical vein endothelial cells described in Example 2. The numbers in parentheses indicate PDL.

FIG. 4 shows the amount of von Willebrand factor produced by the subcultured human umbilical vein endothelial cells described in Example 2.

FIG. 5 shows a comparison of the amount of von Willebrand factor produced by static culture and spinner-cultured vascular endothelial cells shown in Example 3.

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Claims (1)

[Claims] 1. Adhering onto a microcarrier, 20
A method for producing von Willebrand factor, which comprises culturing vascular endothelial cells while applying shear stress in the range of 130 dynes / cm ² .