某皂苷类样品

类器官（Organoids）是指将成体干细胞或多能干细胞在体外三维培养形成的具有一定空间结构的组织类似物。类器官在组织结构、细胞类型、自我更新能力和功能等方面与来源组织高度一致，从而在发育生物学、疾病造模、精准医学、药物研发、基因和细胞疗法、感染和免疫以及再生医学等生物医学的多个领域展现出独特的优势。产品介绍bioGenousTMMouse Intestinal Organoid Kit（小鼠小肠类器官试剂盒）是一款用于建立和维持小鼠肠道成体干细胞来源小肠类器官的完全培养基。生长在该完全培养基中的小鼠小肠类器官主要由干细胞、肠祖细胞和肠绒毛细胞、潘氏细胞、杯状细胞和少量肠内分泌细胞组成，在自我更新能力、组织结构、细胞类型和功能方面，小鼠小肠类器官重现了体内小肠上皮的特征，因此是肠道稳态和疾病机制研究的理想体外模型。技术参数产品信息:产品组成货号体积储存条件及周期bioGenousTMMouse Intestinal Organoid Basal MediumK2001-MI-A100/A500100mL/500mL4℃，12个月bioGenousTMMouse Intestinal Organoid Supplement B(50x)K2001-MI-B100/B5002mL/10mL-20℃,避免反复冻融，12个月bioGenousTMMouse IntestinalOrganoid Supplement C(250x)K2001-MI-C100/C5000.4mL/2mL-20℃,避免反复冻融，12个月EDTA (0.5M, pH 8.0)E2191210.2mL/1mL15-30℃，5年小鼠小肠类器官完全培养基的制备：使用无菌操作技术配制小鼠小肠类器官完全培养基。以下是准备10mL完全培养基的示例，如所需量不同，可相应调整用量。1.冰上解冻Mouse Intestinal Organoid Supplement B (50x)和Mouse Intestinal Organoid Supplement C (250x)。注意：解冻后，建议将Mouse Intestinal Organoid Supplement B (50x)和Mouse Intestinal Organoid Supplement C (250x)分别分装后保存取用，避免反复冻融。2.将200uLMouse Intestinal Organoid Supplement B (50x)，40uLMouse Intestinal Organoid Supplement C (250x)加至9.76mLMouse Intestinal Organoid Basal Medium中，充分混合，配制成10mL小鼠小肠类器官完全培养基。注意：配制后的小鼠小肠类器官完全培养基可在2-8°C储存，建议在两周内使用。Mouse Intestinal Organoid Supplement B(50x)内含有细菌及真菌抗生素(50x)。小鼠小肠类器官原代培养1.依据所在单位批准的实验动物伦理及操作规范牺牲小鼠。2.准备若干培养皿，加入4℃预冷的DPBS备用。3.标准手术操作取小鼠小肠，根据实验需求取总长度约3厘米至20厘米的小肠段，置于含DPBS的培养皿中。4.使用移液管或者注射器往小肠一端注入DPBS以冲洗肠内容物，冲洗后置于新的含DPBS的培养皿中，重复冲洗数次至内容物完全被冲洗干净，置于新的含DPBS的培养皿中。5.使用手术剪将肠管剪开，肠腔面朝上，一只手使用手术镊夹住肠组织一端，另一只手使用手术刀片轻轻刮去肠腔表面肠绒毛，待肠绒毛被刮净后，将肠组织置于新的含DPBS的培养皿中清洗，重复清洗一次。6.将清洗后的小肠组织剪碎至2mm宽，并转移至含有5mmol/L EDTA的预冷DPBS中消化，置于4℃孵育30min。7.消化完成后，将组织碎片转移到新的含DPBS的培养皿中清洗，重复一次以去除EDTA。8.用5mL移液管在含冷的DPBS的培养皿或50mL离心管中吹打、重悬组织碎片，使组织反复穿过移液管尖以产生机械剪切力从而使隐窝与基底层分离，取一部分悬液镜检，当可以看到大量的隐窝样结构后，停止吹打，并对吹打后的组织悬液进行70μm滤网过滤。9.收集穿过滤网的组织悬液，150g离心力4℃离心3min。10.弃上清，使用1mLDPBS重悬组织沉淀，取20uL悬液进行镜检和隐窝计数，计数完成后吸取包含所需隐窝量的悬液，150g离心力4℃离心3min，弃上清后置于冰上。11.用适量的Matrigel重悬组织沉淀，推荐重悬密度为每10uLMatrigel悬液包含200至600个隐窝，重悬后置于冰上，重悬时间不超过30s以避免Matrigel过早凝固。注意：Matrigel稀释比例应在70%以上以保证培养过程中Matrigel的结构稳定性。12.将Matrigel和组织细胞的混合悬液点入24孔板底部正中央，每孔30uL左右，避免悬液接触孔板侧壁。注意：为防止Matrigel室温凝固，此步骤应尽快完成。13.将接种完成后的培养板至于37℃二氧化碳恒温培养箱中，孵育15min左右待Matrigel凝固。14.配制小鼠小肠类器官完全培养基。15.待Matrigel完全凝固后，加入已配制好的小鼠小肠类器官完全培养基，24孔板每孔500uL。注意：请沿壁缓慢加入，避免破坏已凝固结构。16.将24孔板置于37℃二氧化碳培养箱中培养。17.每3天更换一次培养基，更换培养基时应避免破坏Matrigel。18.密切监测类器官生长状态，理想情况下，小鼠小肠类器官应在5至7天内建成。小鼠小肠类器官传代培养19.用经过Anti-Adherence Rinsing Kit(E238002)润洗的枪头吹打刮取类器官，并将类器官和培养基的悬液转移至经过Anti-Adherence Rinsing Kit润洗的1.5mL EP管中。20.用经过Anti-Adherence Rinsing Kit润洗的枪头用力重悬类器官悬浮液，使得类器官与Matrigel分离。21.150g离心力4℃离心3min，弃上清，使用DPBS重悬底部类器官沉淀，150g离心力4℃再次离心3min，弃上清后置于冰上。22.用适量的Matrigel重悬类器官沉淀，重悬后置于冰上，重悬时间不超过30s以避免Matrigel过早凝固。注意：Matrigel稀释比例应在70%以上以保证培养过程中Matrigel的结构稳定性。23.将Matrigel和类器官的混合悬液点入24孔板底部正中央，避免悬液接触孔板侧壁，每孔30uL左右。注意：为防止Matrigel室温凝固，此步骤应尽快完成。24.将接种完成后的培养板至于37℃二氧化碳恒温培养箱中，孵育15min左右待Matrigel凝固。25.配制小鼠小肠类器官完全培养基。26.待Matrigel完全凝固后，加入已配制好的小鼠小肠类器官完全培养基，24孔板每孔500uL。27.将24孔板置于37℃二氧化碳培养箱中培养。

类器官（Organoids）是指将成体干细胞或多能干细胞在体外三维培养形成的具有一定空间结构的组织类似物。类器官在组织结构、细胞类型、自我更新能力和功能等方面与来源组织高度一致，从而在发育生物学、疾病造模、精准医学、药物研发、基因和细胞疗法、感染和免疫以及再生医学等生物医学的多个领域展现出独特的优势。产品介绍Product Description:bioGenousTMMouse Liver Ductal Organoid Kit is a chemically defined cell culture medium for establishment and maintenance of Mouse ductal organoids(mLDs) derived from adult stem cells. Self-renewal of the ductal epithelium is driven by the proliferation of stem cells and their progenitors located in liver. mLDs display all hallmarks of the ductal epithelium in terms of architecture, cell type composition, and self-renewal dynamics, therefore hold great promise for unprecedented studies of Mouse liver development and disease, mLDs may also have applications in regenerative biology through ex vivo expansion of the ductal epithelia.技术参数Product Information:ComponentComponent Cat#VolumeStorage& StabilitybioGenousTMMouse Liver Ductal Organoid Basal Medium（Expansion）K2006-MLD -A100/A500100mL/500mL4℃，12 monthsbioGenousTMMouse Liver Ductal Organoid Supplement B(50x)（Expansion）K2006-MLD –B100/B5002mL/10mL-20℃,avoid repeated freeze-thaw cycles, 12 monthsbioGenousTMMouse Liver Ductal Organoid Supplement C(250x)（Expansion）K2006-MLD –C100/C5000.4mL/2mL-20℃, avoid repeated freeze-thaw cycles, 12 monthsPreparation of Mouse Liver Ductal Organoid Expansion Medium and Maintenance MediumUse sterile technique to prepare the mouse liver ductal organoid expansion medium and maintenance medium. mLDs grown in Mouse Liver Ductal Organoid Expansion Medium overwhelmingly consisted of cholangiocytes. The following examples are for preparing 10 mL of Expansion Medium and Maintenance Medium. If preparing other volumes, adjust accordingly.1.Thaw Mouse Liver Ductal Organoid Supplement B(50x) (Expansion), Mouse Liver Ductal Organoid Supplement C(250x) (Expansion) on ice. Mix thoroughly.NOTE:Once thawed, use immediately or aliquot and store at -20°C for not more than 10 months. After thawing the aliquots, use immediately. Do not re-freeze.2.For Mouse Liver Ductal Organoid Expansion Medium. Add 200 μL Mouse Liver Ductal Organoid Supplement B(50x)(Expansion), 40 μL Mouse Liver Ductal Organoid Supplement C(250x) (Expansion) to 10 mL Mouse Liver Ductal Organoid Basal Medium (Expansion). Mix thoroughly.NOTE:If not use immediately, store complete medium at 2-8°C for not more than 2 weeks. bioGenousTMMouse Liver Ductal Organoid Supplement B (Expansion) contains fungicide and antibiotics(50x).Protocol for Establishment of Mouse Liver Ductal OrganoidsEstablishment of Organoids from Primary Tissue1.Collect primary Mouse liver tissue pieces in ice-cold Primary Tissue Storage Solution (K601005) with conical tubes. Keep tissue samples at 4°C until the start of the isolation.2.Rinse the liver tissuewith Epithelial Organoid Basal Medium(B213151) or DPBSuntil the supernatant is clear.3.Before ducts isolation, thaw Matrigel on ice and keep it cold. Add 5 mL of FBS to 45 mL of Epithelial Organoid Basal Medium to prepare 10% (vol/vol) FBS medium.4.Mince the tissue into small fragments of 5 mm3in a cell culture dish using surgical scissors or scalpels.CRITICALThe dissected samples must be small enough to pass through the tip of a 10 mL pipette.5.Place the dissected pieces of sample into a 15 mL concial tube containing 10 mL of cold Epithelial Organoid Basal Medium with 1% FBS.6.Wash the samples by pipetting with a 10 mL pipette at least ten times.CRITICALFor the subsequent steps, coat the inner surface of every 10 mL pipette with 10% (vol/vol) FBS medium before use to avoid adherence of the samples on the pipette wall.7.Stand the tube still until the samples settle at the bottom. Aspirate the supernatant with a 10 mL pipette and add 10 mL of pre-warmed Tissue Digestion Solution (K601008).8.Digest the tissue fractions in 37℃with rotation at the speed of 200 rpm. The digestion time should not exceed 30 mins.CRITICALTo prevent over-digestion, one should examine under the microscope if the duct structure appear during digestion.9.Once the duct structure appears, stop digestion by transfer the digestion solution into 50mL tube and add 40 ml Epithelial Organoid Basal Medium with 1% FBS.10.Stand the tube for 2 min. Transfer the supernatant into a new tube.11.Spin the supernatant at 4°C at 300g for 3 min. Aspirate and discard the supernatant.12.Re-suspend the pellet with 10 ml Epithelial Organoid Basal Medium with 1%FBS, and spin at 4°C at 300g for 3 min.13.Repeat last step for 2 times.14.Aspirate the supernatant and resuspend the pellet in Matrigel. Matrigel should be kept on ice to prevent it from solidifying thus, work quickly. The amount of Matrigel depends on the size of the pellet. Approximately 20-100 ducts should be plated in 25 μL of Matrigel.CRITICALDo not dilute Matrigel too much (Matrigel should be 70% (Matrigel vol/Total vol)) to ensure formation of solid droplets.15.Plate the Matrigel containing organoids on the bottom of 24-well cell culture plates in droplets of ~30 μL each around the center of the well.CRITICALOnce the organoids are resuspended in Matrigel, proceed with plating as quickly as possible, as the Matrigel may solidify in the tube or pipette tip. Do not let the Matrigel touch the wall of wells.16.Place the culture plate into a humidified incubator at 37 °C and 5% (vol/vol) CO2for15-25 minto let the Matrigel solidify.17.Prepare the required amount of bioGenousTMMouse Liver Ductal Organoid Expansion Medium.18.Once the Matrigel droplets are solidified (15-25 min), open the plate and carefully add 500 μL of Organoid Complete Medium to each well.CRITICALDo not add the medium directly on top of the Matrigel droplets, as this might disrupt the droplets.19.Place the culture plate in a humidified incubator at 37 °C and 5% (vol/vol) CO2.20.Change the medium every 3 days by carefully aspirating medium from the wells and replacing it with fresh, pre-warmed Organoid Complete Medium.21.Closely monitor the organoid formation. Ideally, Mouse Liver Ductal Organoids should be passaged for the first time between 5 and 8 days after initial plating.Splitting and Passaging of Organoids22.Pipette up and down to disrupt the Matrigel, and transfer the organoid suspension into a 1.5 mL conical tube.23.Pipette the organoid suspension up and down to mix thoroughly.Use the bottom of the tube to create pressure, which will aid the removal of Matrigel.24.Centrifuge organoids at 200g for 3 min at room temperature.25.Aspirate the supernatant, and split organoids using either mechanical disruption or Organoid Dissociation Solution (E238001). For mechanical disruption, resuspend the pellet in 1 mL of Organoid Basal Medium. Use a pipette tip to pipette the organoid suspension up and down 30 times. Use the bottom of the tube to create pressure, which will aid organoid disruption. In case of Organoid Dissociation Solution-based cell dissociation, resuspend the pellet in 0.2 mL of Organoid Dissociation Solution, pipette up and down and incubate at 37 °C until organoids fall apart. Pipette up and down with a filter tip for ≥10 times every 1 min to aid in the disruption of the organoids. Monitor digestion closely to keep the incubation time inOrganoid Dissociation Solution to a minimum.CRITICALDo not dissociate in Organoid Dissociation Solution for 3 min, as this may result in poor organoid outgrowth or even loss of the culture. As a rule of thumb, digestion is complete if a mixture of small lumps of cells (consisting of 10–50 cells) can be observed.26.After shearing is complete, wash once by topping up with 1 mL ofOrganoid Basal Medium, and centrifuge at 200g for 3 min at room temperature.27.Aspirate the supernatant and resuspend the organoid pellet in 70% (vol/vol) Matrigel, and plate organoids in droplets on the bottom of a culture plate as describedin Steps 12. After plating is complete, transfer the plate into ahumidified incubator at 37 °C and 5% (vol/vol) CO2for 15–25 min.28.Pre-warm Mouse Liver Ductal Organoid Maintenance Medium at 37 °C.29.After the Matrigel droplets have solidified (15–25 min), carefully pipette pre-warmed medium into the wells.30.Place culture plates in a humidified incubator at 37 °C and 5% (vol/vol) CO2until the organoids are needed for further experiments.

类器官（Organoids）是指将成体干细胞或多能干细胞在体外三维培养形成的具有一定空间结构的组织类似物。类器官在组织结构、细胞类型、自我更新能力和功能等方面与来源组织高度一致，从而在发育生物学、疾病造模、精准医学、药物研发、基因和细胞疗法、感染和免疫以及再生医学等生物医学的多个领域展现出独特的优势。产品介绍Product Description:bioGenousTMMouse Liver Ductal Organoid Kit is a chemically defined cell culture medium for establishment and maintenance of Mouse ductal organoids(mLDs) derived from adult stem cells. Self-renewal of the ductal epithelium is driven by the proliferation of stem cells and their progenitors located in liver. mLDs display all hallmarks of the ductal epithelium in terms of architecture, cell type composition, and self-renewal dynamics, therefore hold great promise for unprecedented studies of Mouse liver development and disease, mLDs could differentiate into hepatocyte like cell under the induction of differentiation medium.技术参数Product Information:ComponentComponent Cat#VolumeStorage& StabilitybioGenousTMMouse Liver Ductal Organoid Basal Medium（Differentiation）K2006-MLH –A100/A500100mL/500mL4℃，12 monthsbioGenousTMMouse Liver Ductal Organoid Supplement B(50x)（Differentiation）K2006-MLH –B100/B5002mL/10mL-20℃,avoid repeated freeze-thaw cycles, 12 monthsbioGenousTMMouse Liver Ductal Organoid Supplement C(250x)（Differentiation）K2006-MLH –C100/C5000.4mL/2mL-20℃, avoid repeated freeze-thaw cycles, 12 monthsbioGenousTMMouse Liver Ductal Organoid Supplement D(250x)（Differentiation）K2006-MLH –D100/D5000.4mL/2mL-20℃, avoid repeated freeze-thaw cycles, 12 monthsPreparation of Mouse Liver Ductal Organoid Differentiation MediumUse sterile technique to prepare the mouse liver ductal organoid differentiation medium. mLDs grown in Mouse Liver Ductal Organoid Expansion Medium overwhelmingly consisted of cholangiocytes. After changing the Expansion Medium to differentiation medium, the mLDs could differentiate into hepatocyte like cells, which display the markers of hepatocytes, including Albumin, Ttr, Cyp3a11 and Mup20. The following examples are for preparing 10 mL of Differentiation I Medium and Differentiation II Medium. If preparing other volumes, adjust accordingly.1.Thaw Mouse Liver Ductal Organoid Supplement B(50x) (Differentiation), Mouse Liver Ductal Organoid Supplement C(250x) (Differentiation) and Mouse Liver Ductal Organoid Supplement D(250x) (Differentiation) on ice.NOTE:Once thawed, use immediately or aliquot and store at -20°C for no more than 10 months. After thawing the aliquots, use immediately. Do not re-freeze.2.For Mouse Liver Ductal Organoid Differentiation Medium I. Add 200 μL Mouse Liver Ductal Organoid Supplement B(50x)(Differentiation), 40 μL Mouse Liver Ductal Organoid Supplement C(250x)(Differentiation) to 10 mL Mouse Liver Ductal Organoid Basal Medium (Differentiation). Mix thoroughly.3.For Mouse Liver Ductal Organoid Differentiation Medium II. Add 200 μL Mouse Liver Ductal Organoid Supplement B(50x)（Differentiation）and 40 μL Mouse Liver Ductal Organoid Supplement C(250x)(Differentiation) and 10 μL Mouse Liver Ductal Organoid Supplement D (250x)(Differentiation) to 10 mL Mouse Liver Ductal Organoid Basal Medium (Differentiation). Mix thoroughly.NOTE:If not use immediately, store complete medium at 2-8°C for no more than 2 weeks. bioGenousTMMouse Liver Ductal Organoid Supplement B (Differentiation) contains fungicide and antibiotics(50x).Protocol for Mouse Liver Ductal Organoids Differentiation1.Culture the mouse ductal organoids in Mouse Liver Ductal Organoid Medium(Expansion) (K2006-MLD) for at least 3 days.2.Change the medium to Mouse Liver Ductal Organoid Differentiation Medium I, and culture for 9 days. During this period, replace the medium every day.3.Change the medium to Mouse Liver Ductal Organoid Differentiation Medium II, and culture for 3 days. During this period, replace the medium every day.4.At the end of this period, the differentiation process is completed. The ductal organoid would differentiate into hepatocyte-like organoid with the expression of hepatic markers, such as Albumin, Ttr, Cyp3a11 and Mup20.