细胞冷冻保存的具体操作步骤

<p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">细胞冷冻保留的具体操作过程</span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">依据技术部给出的最新细胞冷冻保留办法，现公布于下：</span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">1. <font face="宋体">注意事项：</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">1.1. <font face="宋体">欲冷冻保留之细胞应在成长杰出</font><font face="Calibri">(log phase) </font><font face="宋体">且存活率高之状况，约为</font><font face="Calibri">80 </font><font face="宋体">&ndash; </font><font face="Calibri">90 %</font><font face="宋体">致密度。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">1.2. <font face="宋体">冷冻前检查细胞是不是仍保有其特有性质，例如</font><font face="Calibri">hybridoma </font><font face="宋体">应在冷冻保留前一至二日测验是不是有抗体之发生。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">1.3. <font face="宋体">留意冷冻保护剂之质量。</font><font face="Calibri">DMSO </font><font face="宋体">应为试剂级等级，无菌且无色</font><font face="Calibri">(</font><font face="宋体">以</font><font face="Calibri">0.22 micronFGLP Telflon </font><font face="宋体">过滤或是直接购买无菌商品，如</font><font face="Calibri">Sigma D-2650)</font><font face="宋体">，以</font><font face="Calibri">5~10 ml </font><font face="宋体">小体积分装，</font><font face="Calibri">4</font><font face="宋体">℃避光保留，勿作多次冻结。</font><font face="Calibri">Glycerol </font><font face="宋体">亦应为试剂级等级，以高压蒸汽灭菌后避光保留。在敞开后一年内运用，因长时间贮存后对细胞会有毒性。 </font><font face="Calibri">1.4. </font><font face="宋体">冷冻保留之细胞浓度：</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">1.4.1. normal human fibroblast: 1~3 x 106 cells/ml 1.4.2. hybridoma: 1~3 x 106cells/ml<font face="宋体">，细胞浓度不要太高，某些</font><font face="Calibri">hybridoma </font><font face="宋体">会因冷冻浓度太高而在冻结</font><font face="Calibri">24 </font><font face="宋体">小时后死去。 </font><font face="Calibri">1.4.3. adherent tumor lines: 5~7 x 106</font><font face="宋体">，依细胞品种而异。</font><font face="Calibri">Adenocarcinoma </font><font face="宋体">冻结后须较高之浓度，而</font><font face="Calibri">HeLa </font><font face="宋体">只需</font><font face="Calibri">1-3 x 106cells/ml</font><font face="宋体">。 </font><font face="Calibri">1.4.4. other suspensions: 5~10 x 106cells/ml, human lymphocyte </font><font face="宋体">须最少</font><font face="Calibri">5 x 106cells/ml</font><font face="宋体">。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">1.5. <font face="宋体">冷冻保护剂浓度为</font><font face="Calibri">5 </font><font face="宋体">或</font><font face="Calibri">10 % DMSO</font><font face="宋体">，如果不确定细胞之冷冻条件，在做冷冻保留之一起，亦应作一个</font><font face="Calibri">backup culture</font><font face="宋体">，以避免冷冻失利。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">1.6. <font face="宋体">冷冻办法： </font><font face="Calibri">1.6.1. </font><font face="宋体">传统办法</font><font face="Calibri">: 4</font><font face="宋体">℃ </font><font face="Calibri">10 </font><font face="宋体">分钟</font><font face="Calibri">---&gt; -20</font><font face="宋体">℃ </font><font face="Calibri">30 </font><font face="宋体">分钟</font><font face="Calibri">---&gt; -80</font><font face="宋体">℃ </font><font face="Calibri">16 - 18 </font><font face="宋体">小时</font><font face="Calibri">(</font><font face="宋体">或过夜</font><font face="Calibri">) ---&gt; </font><font face="宋体">液氮槽</font><font face="Calibri">vapor phase </font><font face="宋体">长时间贮存。 </font><font face="Calibri">1.6.2. </font><font face="宋体">程序降温：运用等速降温机以&ndash;</font><font face="Calibri">1 </font><font face="宋体">～ </font><font face="Calibri">-3</font><font face="宋体">℃</font><font face="Calibri">/</font><font face="宋体">分钟之速度由室温降至&ndash;</font><font face="Calibri">120</font><font face="宋体">℃，放在液氮槽</font><font face="Calibri">vapor phase </font><font face="宋体">长时间贮存。适用于悬浮型细胞与</font><font face="Calibri">hybridoma </font><font face="宋体">之保留。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">细胞冷冻保留</span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">2. <font face="宋体">资料： </font><font face="Calibri">2.1. </font><font face="宋体">成长杰出之培育细胞 </font><font face="Calibri">2.2. </font><font face="宋体">新鲜培育基 </font><font face="Calibri">2.3. DMSO (Sigma D-2650) 2.4. </font><font face="宋体">无菌塑料冷冻保留管</font><font face="Calibri">(Nalgene 5000-0020) 2.5. 0.4 % w/v trypan blue (GibcoBRL 15250-061) 2.6. </font><font face="宋体">血球计数盘与盖玻片 </font><font face="Calibri">2.7. </font><font face="宋体">等速降温机</font><font face="Calibri">(KRYO 10 Series II)</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">3. <font face="宋体">过程： </font><font face="Calibri">3.1. </font><font face="宋体">冷冻前一日前替换半量或全量培育基，调查细胞成长景象。 </font><font face="Calibri">3.2. </font><font face="宋体">制造冷冻保留溶液</font><font face="Calibri">(</font><font face="宋体">运用前制造</font><font face="Calibri">)</font><font face="宋体">：将</font><font face="Calibri">DMSO </font><font face="宋体">参加新鲜培育基中，最终浓度为</font><font face="Calibri">5-10%</font><font face="宋体">，混合均匀，置于室温下待用。 </font><font face="Calibri">3.3. </font><font face="宋体">依细胞继代培育之操作，搜集培育之细胞，取少数细胞悬浮液</font><font face="Calibri">(</font><font face="宋体">约</font><font face="Calibri">0.1 ml) </font><font face="宋体">计数细胞浓度及冻前存活率。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">3.4. <font face="宋体">离心，去掉上清液，参加适量冷冻保留溶液，使细胞浓度为</font><font face="Calibri">1-5 x 106 cells/ml</font><font face="宋体">，混合均匀，分装于已标明彻底之冷冻保留管中，</font><font face="Calibri">1 ml/vial</font><font face="宋体">，并取少数细胞悬浮液作污染检查。 </font><font face="Calibri">3.5. </font><font face="宋体">冷冻保留办法</font><font face="Calibri">1: </font><font face="宋体">冷冻管置于</font><font face="Calibri">4</font><font face="宋体">℃ </font><font face="Calibri">10 </font><font face="宋体">分钟&rarr; </font><font face="Calibri">-20</font><font face="宋体">℃ </font><font face="Calibri">30 </font><font face="宋体">分钟&rarr; </font><font face="Calibri">-80</font><font face="宋体">℃ </font><font face="Calibri">16</font><font face="宋体">～</font><font face="Calibri">18 </font><font face="宋体">小时</font><font face="Calibri">(</font><font face="宋体">或过夜</font><font face="Calibri">) </font><font face="宋体">&rarr; 液氮槽</font><font face="Calibri">vapor phase </font><font face="宋体">长时间贮存。 </font><font face="Calibri">3.6. </font><font face="宋体">冷冻保留办法</font><font face="Calibri">2: </font><font face="宋体">冷冻管置于已设定程序之等速降温机中，再放入液氮槽中。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">十二月新品优惠中，期待来电咨询订货！</span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p>