Culture medium composition, and method for culturing cell or tissue using said composition
A medium and composition technology, applied in general culture methods, animal cells, cell culture medium, etc., can solve the problems of complicated operation of cell culture in culture vessels, reduced cell viability and reproducibility, etc.
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[0287] (Analysis Example 1: Viscosity measurement and cell suspension test of medium containing deacylated gellan gum)
[0288] Preparation and viscosity measurement of culture medium containing deacylated gellan gum
[0289] Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in pure water to 0.4% (w / v), and dissolved by heating and stirring at 90°C. Allow the aqueous solution to cool to room temperature while stirring, and sterilize in an autoclave at 121°C for 20 min. Place 2 times the concentration of DMEM / F-12 medium (manufactured by Aldrich, 50 mL) and sterile water (47.5 mL) in a 300 mL high beaker, and pass through a homomixer at room temperature (3000 rpm) ) With stirring, the aqueous deacylated gellan gum solution (2.5 mL) was added, and the mixture was continuously stirred for 1 min to prepare a deacylated gellan gum medium composition with a final concentration of 0.01%. A medium composition supplemented with an aqueous deacylated ge...
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[0292] (Comparative example) Preparation of medium containing methylcellulose and collagen
[0293] Preparation of medium containing methylcellulose
[0294] DMEM / F-12 medium (manufactured by Aldrich, 100 mL) was placed in a 200 mL pear-shaped bottle, and methylcellulose (M0387, manufactured by Aldrich, 0.1 g) was added. The mixture was stirred while cooling on an ice bath to dissolve the methyl cellulose. Using this solution, a medium composition in which an aqueous methylcellulose solution was added at a final concentration of 0.1, 0.3, 0.6, or 1.0% (w / v) was prepared.
[0295] Preparation of collagen-containing medium
[0296] Add 10-fold concentration of DMEM / F-12 medium (manufactured by Aldrich, 1 mL), buffer for reconstitution (manufactured by Nitta Gelatin Inc., 1 mL), and pure water (1.5 mL) to 0.3% Type IA cell matrix (manufactured by Nitta Gelatin Inc., 6.5 mL), and the mixture was stirred on ice to generate a medium containing 0.2% collagen. Similarly, a medium compositi...
Example
[0303] (Experimental Example 1: Cell proliferation test by scattered single cells)
[0304] Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w / v), and stirred by heating at 90°C Come to dissolve. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Use this solution by adding deacylated gellan gum at a final concentration of 0.015% (w / v) to IMDM containing 10% (v / v) fetal bovine serum and 10 ng / mL thrombopoietin (manufactured by WAKO) Medium (manufactured by Gibco) to prepare the medium composition. Immediately afterwards, the human leukemia cell line UT7 / TPO was placed in the medium composition supplemented with the above-mentioned deacylated gellan gum to 20,000 cells / mL, and 5 mL / well was dispersed on a 6-well flat-bottomed microculture plate (from Corning Incorporated). Similarly, the human cervical cancer cell line HeLa was spread at 20000 cells / mL by adding 0.015% (...
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