JP2990789B2 - Culture method of vascular endothelial cells - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for culturing vascular endothelial cells for efficiently producing a physiologically active substance derived from vascular endothelial cells.

[Background Art] Vascular endothelial cells (EC) are cells constituting the innermost layer of the inner wall of blood vessels, which control the permeation between blood and tissues, and inhibit many blood clotting, hematopoiesis, blood pressure, etc. It produces active substances (colony stimulating factors, interleukins, endothelins, prostaglandins, etc.) and is deeply involved in the maintenance of living organisms. In addition, proliferation of these cells is an essential factor for repairing damaged blood vessels and angiogenesis in tissues. In order to transfer human ECs removed from a living body to an in vitro culture system, they are different from those derived from bovine or porcine, because of their growth, growth stimulating factor (ECGF) and gelatin (Folkman, J., e.
t al., Proc. Natl. Acad. Sci. USA, 76 , 5217-5221, 197
9), fibronectin (Maciag, T., et al., J. Cell. Bio
l., 91 , 420-426, 1981), collagen (Hoshi, H., et a).
l., Proc. Natl. Acad. Sci. USA, 81 , 6413-6417, 1984)
Requires extracellular matrix components such as Several specific or non-specific growth factors have already been reported (Gospodarowicz, D., Nature, 249 , 123-127, 197).
4), (Shing, Y., et al., Science, 223 , 1296-1299, 198)
4), (Miyazono, K., et al., Biochem. Biophysics. Res. Com.
m., 126 , 83-88, 1985), due to the problems of growth supporting activity and price, it is almost impossible to apply it to large-scale culture. For the purpose of producing substances or using cells per se for artificial blood vessels, The cultivation of human EC in large quantities has never been realized. In addition, since EC has many functions in vivo as described above, maintaining the functionality of cells grown in an in vitro culture system is an important issue. Even with the successful combination of already reported growth factors with the appropriate matrix,
At present, vascular endothelial cell function-preserving culture is limited, and maintaining the function of in vitro culture of human 2-medium fibroblasts is extremely difficult to compare.

[Problems to be Solved by the Invention] An object of the present invention is to culture a large amount of vascular endothelial cells that produce useful physiologically active substances.

[Means for Solving the Problems] The above object is achieved by the present invention described below. That is, in the present invention, when culturing vascular endothelial cells on a microcarrier, the shearing of the culture solution is caused by shear stress of 10 to 130 dyn.
This is a culture method characterized by being in the range of e / cm ² .

Heretofore, adhesion-dependent cells have been cultured on a glass surface or in a rotary culture using a roller bottle because of their adhesion dependence, and thus it has been difficult to mass-cultivate them. But,
In recent years, a microcarrier culture method has been developed, and it has become possible to culture adhesion-dependent cells on a large scale. The present inventors have established a microcarrier culture system for mammalian cells, and have already succeeded in mass-culturing human 2-medium fibroblasts (Sano, E., et al., Cell. Struct. Funct. ., 12 , 509-517,19
87). Using this microcarrier culture system, we tried to grow and mass-produce vascular endothelial cells. For cell growth and production of useful physiologically active substances, appropriate shears depending on the agitation speed is important. I found it to be a point. That is, when growing vascular endothelial cells on a microcarrier, and when producing a physiologically active substance from the grown endothelial cells, shear stress due to stirring of the microcarrier culture is 10 to 130 dyne / cm ² , preferably 20 to 110 dyne. / cm ² range. The shear stress described here is obtained by the following method.

Shear stress (dyne / cm ² ) = average shear rate × viscosity (centi-poise) FIG. 1 shows a spinner culture bottle for microcarrier culture preferably used in the present invention. This is placed on a magnetic stirrer, and the number of revolutions is determined to perform stirring culture. The average shear rate can be obtained from the distance (a) from the tip of the stirrer to the inner wall of the bottle, the inner diameter (b) of the bottle, and the number of revolutions (rpm) according to the following equation.

At this time, since the viscosity of the culture solution is about 1 centi-poise, the shear stress is calculated by displaying the average shear rate in CGS units.

Microcarriers used in the present invention include cross-linked dextran beads to which chemical residues such as diethylaminoethyl groups are bonded, cross-linked dextran beads to which denatured collagen is bonded, polystyrene beads, collagen beads, and gelatin beads whose surfaces are subjected to tissue culture treatment. And preferably crosslinked dextran beads, collagen beads and gelatin beads coated with denatured collagen.

As the vascular endothelial cells, those isolated from the aorta and deficient blood vessels of humans, cows, pigs and the like, human umbilical veins, or vascular endothelial cells transformed by a genetic recombination method are used.

As a culture solution used for culturing vascular endothelial cells, a cell culture medium supplemented with 5 to 20% of serum of animal such as bovine is used, and preferably, M199 medium supplemented with 10% of fetal calf serum is used.

The production of the physiologically active substance is performed using an inducer or without stimulation by the inducer. When an inducer is used, it can be appropriately selected depending on the physiologically active substance to be produced.
inducers such as synthetic or natural RNA such as poly I: poly C, cytokines such as IL-1, TNF, IFNs, or
Growth factors such as PDGF, IGF-β, or PMA, PHA, LP
S, cholera toxin and the like are used.

In the case of human vascular endothelial cells, besides this, basic-FGF,
It is preferable to add vascular endothelial cell growth factor (ECGF) derived from acidic-FGF various tumor cells, normal cells, platelets and the like.

[Examples] Hereinafter, the present invention will be specifically described with reference to examples.

Example 1 Vascular endothelial cells isolated by enzymatic perfusion from human umbilical vein were cultured in a collagen-coated flask in fetal bovine serum.
The cells were cultured and grown in M199 medium supplemented with 10% and 20% of the culture supernatant of human normal diploid fibroblasts containing ECGF activity. 0.
200 ml of the above culture solution was placed in a spinner culture bottle (500 ml capacity) containing 3 w / v% gelatin beads (Gelibead, Hazleton) as shown in FIG.
× 10 ⁵ / ml was inoculated. At 37 ° C, stirring speed is 50 ~
It was set at 600 rpm (11 to 128 dyne / cm ² ) and cultured for 8 days to examine its growth. The results are shown in FIG. This indicated that the proliferation of vascular endothelial cells was dependent on shear stress.

Example 2 Human umbilical vein-derived vascular endothelial cells were cultured in a plane according to Example 1, and inoculated into a spinner culture bottle, and 300 rpm (65 dy)
ne / cm ² ), and the medium was changed once every two days to grow to confluence. Growth-saturated cells should be
After washing with M199 medium, adding the same serum-free medium and culturing at various rotation speeds (shear stress) for 2 days, the amount of 6-ketoprostaglandin F1α produced was measured by radioimmunoassay, and the results are shown in FIG. Indicated. Thus, 6-ketoprostaglandin F1α specifically produced by vascular endothelial cells
It can be seen that production of is regulated depending on shear stress.

Example 3 An experiment was performed in the same manner as in Example 2, and the amount of interleukin-6 (IL-6) produced in a serum-free medium was examined. The results are shown in FIG.

The vascular endothelial cells grown on the microcarrier were washed with M199 medium, cultured at 0 to 300 rpm (0 to 65 dyne / cm ² ) using the same serum-free medium, and IL produced by repeating harvesting every 2 to 3 days -6 was measured using an enzyme immunoassay. This revealed that production of IL-6 was also produced depending on shear stress.

[Effects of the Invention] According to the present invention, mass culture of vascular endothelial cells, which has conventionally been difficult, can be efficiently performed. Therefore, useful bioactive substances produced by vascular endothelial cells can be produced in large quantities.

[Brief description of the drawings]

FIG. 1 shows a culture bottle preferably used in the present invention, wherein a shows the distance from the tip of the stirrer to the inner wall of the bottle, and b shows the inner diameter of the culture bottle. FIG. 2 shows shear stress exerted on the proliferation of vascular endothelial cells in Example 1. FIG. 3 shows the effect of shear stress on the amount of 6-ketoprostaglandin F1α produced by the vascular endothelial cells of Example 2. FIG. 4 shows the effect of shear stress on the amount of IL-6 produced by the vascular endothelial cells of Example 3.

Continuation of front page (56) References Am. J. Physiol. , 259 (3 PART 2), H804-H812 (1990) Clin. Invest. , 79 (2), 600-608 (1987) Translated by Yuji Mitsui, "The Practice of Animal Cell Culture" (Published February 28, 1990) Maruzen Co., Ltd. p. 66, 67 (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 5/06-5/10 BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] (1) When culturing vascular endothelial cells on a microcarrier, shear stress caused by agitation of the culture solution is 20 to 110.
A method for culturing vascular endothelial cells, characterized in that it is in the range of dyne / cm ² .