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【干貨分享】小鼠膽管類器官原代建立Protocol</span> </div> <div class="right_content"> <div class="art-cont"> <h1 class="art-title">【干貨分享】小鼠膽管類器官原代建立Protocol</h1> <div class="art-info"> 發布時間： 2023-07-24　　點擊次數： 280次 </div> <div class="art-body"> <p><br style="text-align: left; margin-bottom: 10px;"/></p><p style="text-align: left; margin-bottom: 10px;"><br/></p><section data-role="outer" label="edit by 135editor"><section><section><section data-role="paragraph"><section data-tools="135編輯器" data-id="126741"><section><section data-width="100%"><section data-role="paragraph"><p style="text-align:center"><img data-backh="417" data-backw="556" data-ratio="0.75" data-src="https://mmbiz.qpic.cn/sz_mmbiz_jpg/SJyibI36BxE3ZTzBd77ibNe1TWUCkfAS12rxY505icLnL0l3rpicaZjCjgCLX5MHaeHvmLicJaB1iasZjwia1vYSMYKKA/640?wx_fmt=jpeg" data-type="jpeg" data-w="1080" data-index="2" src="https://img68.chem17.com/9/20230724/638257855247633223819.jpg" _width="100%" crossorigin="anonymous" alt="圖片" data-fail="0" style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important; vertical-align: inherit; height: auto !important; width: 655px; visibility: visible !important;"/></p><p style="text-align: center; margin-bottom: 10px;">小鼠膽管類器官</p></section><section data-tools="135編輯器" data-id="127782"><section><section><section><section><section><section><strong><span style="font-size: 16px;">一、實驗目的</span></strong></section></section></section></section><section data-autoskip="1"><p>1.熟練掌握小鼠膽管類器官原代建立技術。 </p><p>2.了解掌握小鼠多種組織來源類器官原代建立技術。 </p><p>3.觀察記錄小鼠膽管原代類器官生長、擴增過程中類器官形態的變化。</p><p><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></p></section></section><section><section><section><section><section><span style="font-size: 16px;"><strong>二、實驗原理</strong></span></section></section></section></section><section data-autoskip="1"><p>本實驗策略采取從成體干細胞中建立和維持小鼠膽管類器官的方法。膽管上皮的自我更新是由位于肝臟的干細胞及其祖細胞的增殖所驅動的。小鼠膽管類器官表達膽管上皮在結構、細胞類型組成和自我更新動力學方面的所有標志，因此為小鼠肝臟發育和疾病的研究提供了巨大的希望。 </p><p><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></p></section></section><section><section><section><section><section><strong><span style="font-size: 16px;">三、實驗儀器、材料、試劑</span></strong></section></section></section></section><section data-autoskip="1"><p>1. 儀器：生物安全柜、細胞培養箱、低溫水平式離心機、倒置顯微鏡、低溫冰箱、搖床。</p><p>2. 材料：細胞培養板(規格為24孔、48孔和96孔)、離心管(規格為1.5mL、15mL和50mL)、細胞培養皿(直徑規格為2.5cm和6cm)、移液器(規格為2.5μL、10μL、20μL、200μL、1000μL和5000μL)、無菌吸頭(規格為10μL、200μL和1000μL)、無菌鑷子、無菌組織剪。</p><p>3.試劑：小鼠膽管類器官培養試劑盒(MA-0817H009HL/S)、組織消化液(MB-0818L06S-100)、潤洗液(MB-0818L03L/S)、基礎培養基(MB-0818L07)、胎牛血清、DPBS、基質膠(082701/082703/082755)、紅細胞裂解液(MB-0818L08L/S)。</p><section><section data-autoskip="1"><p><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></p></section></section><section><section><section><section><section><strong><span style="font-size: 16px;">四、實驗內容與方法</span></strong></section></section></section></section></section></section></section></section></section></section><section data-autoskip="1"><section data-tools="135編輯器" data-id="123360"><section><section><section><section><br/></section><section><span style="font-size: 16px;">01 實驗前準備</span></section></section><section data-width="100%"><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></section><section><section data-autoskip="1"><section data-role="list"><ul class=" list-paddingleft-2" style="list-style-type: square;"><li><p>小鼠膽管類器官培養試劑盒(MA-0817H009HL/S)、基質膠(082701/082703/082755)4℃化凍；</p></li></ul></section><p><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></p><section data-role="list"><ul class=" list-paddingleft-2" style="padding: 0px 0px 0px 30px; outline: 0px; max-width: 100%; list-style-position: outside; box-sizing: border-box !important; overflow-wrap: break-word !important;"><li><p>小鼠膽管類器官培養基的制備：將200μL Mouse Liver Ductal Organoid Supplement B (50x)，40μL Mouse Liver Ductal Organoid Supplement C (250x) 加至9.76mL Mouse Liver Ductal Organoid Basal Medium中，充分混合，配制成10mL小鼠膽管類器官培養基，室溫平衡，可在2-8°C儲存，建議在兩周內使用；</p></li></ul></section><p><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></p><section data-role="list"><ul class=" list-paddingleft-2" style="padding: 0px 0px 0px 30px; outline: 0px; max-width: 100%; list-style-position: outside; box-sizing: border-box !important; overflow-wrap: break-word !important;"><li><p>準備足量添加1%雙抗的DPBS；</p></li></ul></section><section data-role="list"><ul class=" list-paddingleft-2" style="padding: 0px 0px 0px 30px; outline: 0px; max-width: 100%; list-style-position: outside; box-sizing: border-box !important; overflow-wrap: break-word !important;"><li><p>配制10mL組織消化液，現配現用，37℃搖床混勻預熱，未用完消化液可-20℃保存1周（Mogengel：10mL基礎液+添加物500μL)。</p></li></ul></section></section></section></section></section></section><section data-tools="135編輯器" data-id="123360"><section><section><section><section><span style="font-size: 16px;"><br/></span></section><section><span style="font-size: 16px;">02 組織樣本收集</span></section></section><section data-width="100%"><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></section><section><section data-autoskip="1"><section data-role="list"><p>小鼠斷脊處死，表面噴灑酒精殺菌，在無菌條件下將膽管第一大肝葉取出，用刀片切下1/2肝葉，放入4℃預冷的DPBS溶液中。</p></section></section></section></section></section></section><section data-tools="135編輯器" data-id="123360"><section><section><section><section><br/></section><section><span style="font-size: 16px;">03 組織清洗</span></section></section><section data-width="100%"><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></section><section><section data-autoskip="1"><section data-role="list"><p>用冰冷的DPBS清洗2~3次，將組織轉移到新的6cm培養皿或1.5mL EP管中，用無菌組織剪將組織剪成約0.5mm3的碎片。</p></section></section></section></section></section></section><section data-tools="135編輯器" data-id="123360"><section><section><section><section><span style="font-size: 16px;"><br/></span></section><section><span style="font-size: 16px;">04去除雜質細胞</span></section></section><section data-width="100%"><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></section><section><section data-autoskip="1"><section data-role="list"><p>將剪碎的組織轉移到15mL離心管中，并加入約10mL冰冷的Basal medium。用5mL移液管上下吹打樣品，以去除紅血球和脂肪(它們會漂浮到溶液的頂部)。后讓組織碎片沉降5~7分鐘，并棄去約7.5mL的上清液，包括任何漂浮的脂肪，后重復此洗滌步驟1~2次。</p></section></section></section></section></section></section><section data-tools="135編輯器" data-id="123360"><section><section><section><section><br/></section><section>05消化液消化</section></section><section data-width="100%"><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></section><section><section data-autoskip="1"><section data-role="list"><p>將剪碎的組織用適量的原代組織液消化液(MB-0818L06S-100)重懸，并轉移至新的離心管內，于37℃，100rpm條件下的恒溫振蕩培養箱中，消化20-30分鐘，每隔10分鐘觀察組織消化情況，待組織塊明顯分散，懸液較渾濁時，取適量組織消化懸液鏡檢，待懸液中有較多膽管結構即可終止消化，若組織碎塊較大或消化不可適當延長消化時間。消化期間可以使用不同規格的移液器吹打組織消化懸液，幫助充分消化。 </p></section></section></section></section></section></section><section data-tools="135編輯器" data-id="123360"><section><section><section><section><br/></section><section>06終止消化</section></section><section data-width="100%"><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></section><section><section data-autoskip="1"><section data-role="list"><p>在消化完成的組織懸液中加入胎牛血清（Fetal Bovine Serum, FBS）至終濃度達1%~5%以減緩消化作用，同時輕輕吹打5~10次。 </p></section></section></section></section></section></section><section data-tools="135編輯器" data-id="123360"><section><section><section><section>07靜置收集</section></section><section data-width="100%"><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></section><section><section data-autoskip="1"><section data-role="list"><p>靜置3-10min，待組織碎片自然沉降后，將上清液轉移至新的15mL離心管中，剩余組織碎片中加入1mL冰冷的Basal medium吹打5~10次，靜置1min待組織碎片自然沉降后，將上清收集至離心管中。在8℃下以250g的速度離心3min，棄去上清以洗去殘留的消化液。若所獲細胞沉淀呈紅色，說明其中包含紅細胞，則需在清洗前于沉淀中加入紅細胞裂解液(MB-0818L08L/S)裂解紅細胞，并于250g離心力離心3分鐘，吸去上清液保留沉淀，再對沉淀進行清洗。</p></section></section></section></section></section></section><section data-tools="135編輯器" data-id="123360"><section><section><section><section><br/></section><section>08類器官收集</section></section><section data-width="100%"><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></section><section><section data-autoskip="1"><section data-role="list"><p>用含有抗生素的PBS或基礎培養基(MB-0818L07)清洗細胞2次（混勻后250g離心力離心，3分鐘，棄去上清液）以除去FBS。 </p></section></section></section></section></section></section><section data-tools="135編輯器" data-id="123360"><section><section><section><section><br/></section><section>09基質膠3D包裹</section></section><section data-width="100%"><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></section><section><section data-autoskip="1"><section data-role="list"><p>將清洗后的細胞沉淀用含有抗生素的PBS或基礎培養基重懸，取少量懸液進行活細胞檢測和計數，并按照100,000~500,000個細胞/mL的密度加入細胞外基質（>70%）并在冰上混勻，混勻后置于冰上。</p><p><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></p><p>注意：混勻吹打的動作要輕柔，切忌產生大量氣泡，常溫混勻則需要控制在15秒內。</p></section></section></section></section></section></section><section data-tools="135編輯器" data-id="123360"><section><section><section><section><br/></section><section>10 種板培養</section></section><section data-width="100%"><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></section><section><section data-autoskip="1"><section data-role="list"><p>用移液器吸取細胞外基質和細胞的混合液移至細胞培養孔板中，（如24孔細胞培養板每孔點20~30μL混合懸液，48孔板每孔點12.5~18μL混合懸液，96孔板孔板每孔點3~5μL混合懸液），混合懸液須點至培養孔底部中央位置，其鋪開后不可接觸培養孔側壁。將培養板放入37℃，在5%CO2細胞培養箱中孵育20分鐘，待細胞外基質凝固至不再流動后，沿孔壁緩緩加入培養基，(如24孔板加500μL培養基，48孔板加250μL培養基，96孔板加100μL培養基)，P0代培養3~4天換液。 </p></section></section></section></section></section></section><section data-tools="135編輯器" data-id="123360"><section><section><section><section><br/></section><section>11 持續培養</section></section><section data-width="100%"><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></section><section><section data-autoskip="1"><section data-role="list"><p>將細胞培養板放入培養箱中進行培養，每1天于倒置顯微鏡下觀察類器官的生長狀態，每3天于倒置顯微鏡下拍照，記錄多個視野中的類器官的形態和分布。</p></section></section></section></section></section></section><section data-role="paragraph"><p><br style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important;"/></p><section><section><section><section><section><strong><span style="font-size: 16px;">五、小鼠膽管類器官培養推薦試劑</span></strong></section></section></section></section></section><p><img class="rich_pages wxw-img" data-backh="234" data-backw="556" data-ratio="0.4203703703703704" data-src="https://mmbiz.qpic.cn/sz_mmbiz_png/SJyibI36BxE3ZTzBd77ibNe1TWUCkfAS12j26wl2tsEGfn6OLNLz7UpjfDPOrXrQ1Od12BTRAAQSY8yUU1bfPicFQ/640?wx_fmt=png" data-type="png" data-w="1080" data-index="3" src="https://img68.chem17.com/9/20230724/638257855249820823881.png" _width="100%" crossorigin="anonymous" alt="圖片" data-fail="0" style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important; overflow-wrap: break-word !important; vertical-align: inherit; height: auto !important; width: 655px; visibility: visible !important;"/></p></section></section></section></section></section></section></section></section><p><br/></p> </div> <div class="art-next"> <dl class="shangxia"> 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