In situ harvesting of adhered target cells using thermoresponsive substrate under a microscope: principle and instrumentation.
A novel technique and instrumented device were developed to harvest target cells from multicellular mixture of different cell types under a microscope. The principle of the technique is that cells cultured on a thermoresponsive-substance-coated dish were detached by a region-specific cooling device and simultaneously harvested using a micropipette, both of which were assembled in an inverted microscope. Thermoresponsive coating consists of the mixture of poly(N-isopropylacrylamide) (PNIPAAm) and PNIPAAm-grafted gelatin. The former non-cell-adhesive polymer dissolves below at 32.1 degrees C in water and precipitates over that temperature (called lower critical solution temperature, LCST), and the latter cell-adhesive polymer has LCST of 34.1 degrees C. The appropriate mixing ratio of these thermoresponsive polymers exhibited high cell adhesion at physiological temperature and complete cell detachment at room temperature. A device developed as to cool at only a tiny area of the bottom of the dish, beneath which a cell that was targeted under a microscope, was assembled in a microscope. It was demonstrated that single cell or two cells that adhered to each other was detached from the surface and harvested by a micropipette within approximately 30s.
Takamatsu
Hiroshi
H
Department of Mechanical Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan. takamatsu@mech.kyushu-u.ac.jp <takamatsu@mech.kyushu-u.ac.jp>
Uchida
Satoru
S
Matsuda
Takehisa
T
D016475:3T3 Cells; D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods*; D002478:Cells, Cultured; D011232:Chemical Precipitation; D005780:Gelatin / Q000737:chemistry; D051379:Mice; D008858:Microscopy, Phase-Contrast; D008904:Miniaturization / Q000295:instrumentation; D011108:Polymers / Q000737:chemistry; D012995:Solubility; D013499:Surface Properties; D013696:Temperature
D000180:Acrylic Resins; D011108:Polymers; C052970:poly-N-isopropylacrylamide; D005780:Gelatin