In Vivo Scientific, LLC 5 Gybe Ho Ct, Salem, SC, 26976, USA.
CellEraser, LLC 15649 Century Lake Dr., Chesterfield, MO, 63017, USA.
Sci Rep. 2018 Oct 3;8(1):14755. doi: 10.1038/s41598-018-32950-w.
As an alternative to laser-based methods, we developed a novel in situ cell isolation method and instrument based on local water absorption of millimeter wave (MMW) radiation that occurs in cellular material and nearby culture medium while the cultureware materials (plastic and glass) are transparent to MMW frequencies. Unwanted cells within cell population are targeted with MMWs in order to kill them by overheating. The instrument rapidly (within 2-3 seconds) heats a cell culture area of about 500 µm in diameter to 50 °C using a low-power W-band (94 GHz) MMW source. Heated cells in the area detach from the substrate and can be removed by a media change leaving a bare spot. Hence we named the instrument "CellEraser". Quick, local and non-contact heating with sharp boundaries of the heated area allows elimination of the unwanted cells without affecting the neighboring cells. The instrument is implemented as a compact microscope attachment and the selective hyperthermic treatment can be done manually or in an automated mode. Mammalian cells heated even momentarily above 50 °C will not survive. This "temperature of no return" does not compromise cellular membranes nor does it denature proteins. Using the CellEraser instrument we found that the key event that determines the fate of a cell at elevated temperatures is whether or not the selectivity of its nucleus is compromised. If a cell nucleus becomes "leaky" allowing normally excluded (cytoplasmic) proteins in and normally nuclear-localized proteins out, that cell is destined to die. Quick heating by MMWs to higher temperatures (70 °C) denatures cellular proteins but the cells are not able to detach from the substrate - instead they undergo a phenomenon we called "thermofixation": such cells look similar to cells fixed with common chemical fixatives. They remain flat and are not washable from the substrate. Interestingly, their membranes become permeable to DNA dyes and even to antibodies. Thermofixation allows the use of western blot antibodies for immunofluorescence imaging.
作为激光方法的替代方案，我们开发了一种新的基于毫米波及原位细胞分离方法和仪器，该方法基于毫米波辐射在细胞物质和附近培养基中的局部吸收，而培养皿材料（塑料和玻璃）对毫米波频率是透明的。利用毫米波使目标细胞群体中的不需要的细胞过热致死。该仪器使用低功率 W 波段（94GHz）毫米波源，可在 2-3 秒内快速将直径约 500µm 的细胞培养区域加热至 50°C。该区域内的受热细胞从基底上脱离，并可通过更换培养基去除，从而留下裸露区域。因此，我们将该仪器命名为“CellEraser”。快速、局部和非接触加热，加热区域边界清晰，可在不影响相邻细胞的情况下消除不需要的细胞。该仪器作为紧凑型显微镜附件实施，选择性的过热处理可以手动或自动完成。哺乳动物细胞即使瞬间加热到 50°C 以上也无法存活。这种“不归点”既不会损害细胞膜，也不会使蛋白质变性。使用 CellEraser 仪器，我们发现决定细胞在高温下命运的关键事件是其核的选择性是否受到损害。如果细胞核变得“渗漏”，允许通常被排除（细胞质）的蛋白质进入并使通常定位于核内的蛋白质逸出，那么该细胞注定会死亡。毫米波快速加热到更高温度（70°C）会使细胞蛋白质变性，但细胞无法从基底上脱离-相反，它们会经历我们称之为“热固定”的现象：这种细胞类似于用常见化学固定剂固定的细胞。它们保持平坦，不能从基底上洗掉。有趣的是，它们的膜对 DNA 染料甚至抗体变得具有渗透性。热固定允许使用免疫印迹抗体进行免疫荧光成像。
