JPS6231909B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for efficiently growing animal cells in vitro.

The growth of animal cells on artificial substrates such as glass and plastic is well known and has enabled the production of biological products such as vaccines and hormones.

Recently, attempts have been made to use special ion-exchange resin beads (microcarriers) and semipermeable hollow fibers as culture substrates to culture at higher densities or in conditions closer to in vivo conditions.

However, with the exception of special cells such as mouse L cells and Latte's sarcoma cells that can proliferate without adhering to the substrate surface, adhesion and deformation to the substrate surface are important for cell proliferation prior to division and proliferation. essential (adhesion-dependent cell proliferation, anchoring
(effect) Cells that fail to adhere to the surface within a certain period of time will die.

In particular, when using floating culture using the aforementioned microcarriers or a hollow system with a small curvature as a substrate, the probability of contact between cells and the surface of the substrate or the contact time is small, so the amount of initial cell adhesion is small and it is difficult to Cell mass may not grow efficiently.

On the other hand, in order for animal cells to perform normal growth activities, serum components (especially from calves or bovine fetuses) are required in addition to various amino acids, vitamins, sugars, salts, oxygen, etc. is grown and stored in serum-containing medium.

The present inventors conducted extensive studies with the aim of increasing the initial amount of cell adhesion as described above, and as a result, they discovered that serum components essential for cell culture have a large effect on cell adhesion, and thus arrived at the present invention.

That is, the present invention relates to a method for culturing cells, which comprises bringing cells suspended in a medium substantially free of serum into contact with a culture substrate, and then adding serum to the medium.

Serum-free cell suspensions are prepared by removing the medium from a layer of cells growing in serum-containing medium and then adding serum-free medium (e.g., Eagle).
After washing several times with MEM, etc.), trypsin,
It can be obtained by suspending cells detached from the substrate with EDTA or the like in a serum-free medium. The cell suspension thus obtained is brought into contact with the substrate for 0.02 to 10 hours, preferably 0.1 to 5 hours, in a carbon dioxide incubator at 37°C, and then the concentration becomes 5 to 10%. Serum (preferably calf or fetal bovine) is added or the medium is replaced with a medium containing 5-10% serum and culture is continued.

The method of the present invention can be applied to any culture method, including conventional culture methods using glass or plastic dishes or culture bottles, or new methods using semipermeable membranes and the like. In particular, the effects of the present invention are particularly apparent in the microcarrier method and the culture method using hollow fibers.

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

Example 1 Human fetal-derived cells were suspended in serum-free Eagle's medium (1.5 x ¹⁰ cells/ml), and Falcon
3 hours after dispensing 5 ml into a polystyrene sialet.37
Culture in a carbon dioxide incubator at ℃, and replace with Eagle medium containing 5% calf serum.
After culturing for 48 hours, the amount of cell protein was quantified by the Lowry method and found to be 42 μg/cm ² .

On the other hand, when serum was added from the beginning using the same method and conditions, the protein amount was 30 μg/cm ² .

Example 2 Human foreskin-derived cells (Flow7000) were suspended in serum-free Eagle medium (2 x ¹⁰ cells/cell number).
ml), Amicon hollow fiber cell culture device (Vitafiber)
The cells were injected into the fibers and cultured in a carbon dioxide incubator at 37°C for 2 hours, then replaced with Eagle's medium containing 10% calf serum, perfused with fresh medium inside the hollow fibers, and cultured for 1 week. When the amount of proliferating cells was continuously quantified, the amount of cells obtained was 2.3 times that when serum was added from the beginning.

Claims (1)

[Claims] 1. A method for culturing cells, which comprises bringing cells suspended in a medium substantially free of serum into contact with a culture substrate, and then adding serum to the medium.