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View additional product information for Dog (Beagle) Cryopreserved Hepatocytes, plateable Male - FAQs (DGCP10)
24 product FAQs found
这能是测试化合物具有毒性造成的，其它可能的原因和相关建议如下：
- 应用了不合适的培养基：使用Gibco William’s E培养基和Gibco种板与培养添加剂套装；请参考我们的培养方案
- 此肝细胞批次未鉴定可用于贴壁培养：检查本批次产品的说明书以确保其可用于贴壁培养
- 细胞培养时间过长：通常情况下，可种板培养的冻存肝细胞的培养时间不应超过五天
首先推荐您将实验结果与对应批次的特征说明表（人源细胞）中的展示结果进行比较，并推荐您参考我们的酶诱导的操作步骤。此外其他可能的原因和相关建议如下：
- 单层细胞的汇合度不佳：请参考“我的单层肝细胞汇合度不佳。我该如何处理？”的可能原因和建议部分
- 单层细胞的完整性不佳：请参考“我发现所培养的肝细胞聚集，观察到细胞碎片和/或单层细胞出现孔洞，表明这些细胞正在死亡。我该如何处理？”的可能原因和建议部分
- 不适当的阳性对照：检查阳性对照以确保其适用性
- 阳性对照的应用浓度错误：使用正确浓度的阳性对照
请参见下列原因和相关建议：
- 这一批次的肝细胞不是 transporter-qualified 的：检查本批次产品的规格说明书以确保其是transporter-qualified的
- 不合适的培养基：使用Gibco William’s E培养基和Gibco种板与培养添加剂套装；请参考我们的培养方案
- 毛细胆管形成时间不足：通常情况下，至少需要培养4-5天才能形成毛细胆管网络
请参见下列原因和相关建议：
- 此肝细胞批次未鉴定可用于贴壁培养：检查本批次产品的说明书以确保其可用于贴壁培养
- 应用了不合适的培养基：使用Gibco William’s E培养基和Gibco种板与培养添加剂套装；请参考我们的培养方案
- 细胞培养时间过长：通常情况下，可种板培养的冻存肝细胞的培养时间不应超过五天
请参见下列原因和相关建议：
- 接种密度过高：检查特定批次的特征说明表以获取合适的接种密度（人体细胞）的相关信息；培养前在显微镜下镜检，以确保细胞的接种密度合适
- 种板过程中肝细胞不够分散：在培养箱中通过8字和前后模式缓慢晃动培养板来均匀分散细胞；在加入Geltrex Matrix之前振荡平板并润洗单层细胞。
- 培养板中培养基体积不足：请参考文献或咨询技术支持来获得关于培养基体积的建议信息
请参见下列原因和相关建议：
- 接种密度过低：检查特定批次的特征说明表以获取合适的接种密度（人体细胞）的相关信息；培养前在显微镜下镜检，以确保细胞的接种密度合适
- 种板过程中肝细胞不够分散：在培养箱中通过8字和前后模式缓慢晃动培养板来均匀分散细胞
- 培养板中培养基体积不足：请参考文献或咨询技术支持来获得关于培养基体积的建议信息
- 贴壁效率较低:请参见“我的肝细胞贴壁效率低，我该如何处理？”的原因和建议部分
- 一些批次的动物产品的汇合度在80%以下：请参见批次特异的参数信息表以获知合适的接种密度。注意：一些动物种类的细胞会产生细胞链或岛而不能达到100%汇合度。
请参见下列原因和相关建议：
- 不当的复苏操作：浏览复苏，种板和计数实验方案；在37°C化冻细胞的时间应小于2分钟
- 不合适的复苏培养基：在复苏过程中使用HTM培养基来去除冷冻保护剂
- 不正确的离心速度：按复苏方案检查离心速度和时间是否合适（这些条件随细胞的种属来源而变；通常人源细胞可在室温下以100 x g的转速离心10分钟）
- 计数肝细胞时操作过于剧烈：缓慢混匀；使用内径较宽的移液枪头
- 不当的计数操作：请在计数前确保细胞分散均匀；对4组网格线中的两组进行计数；载入计数板前不要让细胞在台盼蓝混合液中停留超过1分钟
请参见下列原因和相关建议：
- 不当的复苏操作：浏览复苏，种板和计数实验方案；在37°C化冻细胞的时间应小于2分钟
- 不合适的复苏培养基：在复苏过程中使用HTM培养基来去除冷冻保护剂
- 计数肝细胞时操作过于剧烈：缓慢混匀；使用内径较宽的移液枪头
- 不当的计数操作：请在计数前确保细胞分散均匀；对4组网格线中的两组进行计数；载入计数板前不要让细胞在台盼蓝混合液中停留超过1分钟
- 细胞在外放置时间过长：计数后立即接种细胞
不同于永生化的细胞系，肝细胞是不能无限期培养的原代细胞。复苏后的悬浮肝细胞仅限于最多4-6小时孵育的短期实验。种板于胶原包被塑料器皿的培养基中的可贴壁肝细胞通常能维持2-7天的代谢活性，实际情况视具体应用而定。
如经适当保存，冻存的肝细胞可在气相液氮（–135°C或更低温度）中保存数年之久。这对于开展长达数月的系列实验是十分理想的选择。
冻存的肝细胞被保存于干式液氮（LN2）气相运输器或杜瓦瓶的无害容器中，通过气相液氮进行运输。杜瓦瓶的内部温度维持在–140°C与–160°C之间，不开启的条件下通常能够保持此温度范围长达7-10天时间。肝细胞一经收到就应移至实验室内长期的LN2罐中，之后将杜瓦瓶归还至预付返货标签上列出的地址以供重复使用。
一旦收到冻存肝细胞，请小心迅速地将冻存管移至气相液氮中，并保持在–135°C以下的温度中直至使用。在开展实验前，冻存管发生任何温度上升都可能会危及肝细胞的活力、功能与细胞活性。
Thermo Fisher Scientific has a dedicated team to answer questions regarding hepatocytes. You can send a question by email (hepaticproducts@thermofisher.com) or contact us by phone: US toll-free: (866) 952 3559
This could be due to the toxicity of the test compound. Here are other potential causes and recommendations:
-Sub-optimal culture medium: Use Williams Medium E with Plating and Incubation Supplement Packs; refer to our plating protocol
-Hepatocyte lot not characterized as plateable: Check lot specifications to ensure it is qualified for plating
-Cells were cultured for too long: In general, plateable cryopreserved hepatocytes should not be cultured for more than five days
First of all, we recommend comparing results to those reported on our lot-specific characterization specification sheet (human cells) and also referring to our enzyme induction protocol. Here are other potential causes and recommendations:
-Sub-optimal monolayer confluency: Please see our recommendations for 'I have a sub-optimal monolayer confluency for my hepatocytes. What should I do?'
-Poor monolayer integrity: Please see our recommendations for 'With my hepatocytes, I’m seeing rounding up of the cells, cellular debris, and/or holes in the monolayer, indicating dying cells. What should I do?'
-Inappropriate positive control: Check positive control to ensure suitability
-Incorrect concentration of positive control: Use the correct concentration of positive control
Please see the following causes and recommendations:
-Hepatocyte lot not transporter-qualified: Check lot specifications to ensure it is transporter-qualified
-Sub-optimal culture medium: Use Williams Medium E with Plating and Incubation Supplement Packs; refer to our plating protocol
-Not enough time for bile canaliculi to form: In general, at least 4-5 days in culture is required for bile canalicular network formation
Please see the following causes and recommendations:
-Hepatocyte lot not characterized as plateable: Check lot specifications to ensure it is qualified for plating
-Sub-optimal culture medium: Use Williams Medium E with Plating and Incubation Supplement Packs; refer to our plating protocol
-Cells were cultured for too long: In general, plateable cryopreserved hepatocytes should not be cultured for more than five days
Please see the following causes and recommendations:
-Seeding density too high: Check lot-specific characterization specification sheet for appropriate seeding density (human cells); observe cells under microscope for appropriate seeding prior to incubation
-Insufficient dispersion of hepatocytes during plating: Disperse cells evenly by moving plate slowly in a figure-eight and back-and-forth pattern in incubator; shake plate and wash cell monolayers prior to applying Geltrex Matrix overlay
-Improper plating volume used for well format : Refer to literature or technical support for suggested plating volumes
Please see the following causes and recommendations:
-Seeding density too low: Check lot-specific characterization specification sheet for appropriate seeding density (human cells); observe cells under microscope for appropriate seeding prior to incubation
-Insufficient dispersion of hepatocytes during plating: Disperse cells evenly by moving plate slowly in a figure-eight and back and forth pattern in incubator
-Insufficient plating volume used for well format: Refer to literature or technical support for suggested plating volumes
-Low attachment efficiency: Please see our recommendations for: 'I'm getting low attachment efficiency with my hepatocytes. What should I do?'
-Some animal lots are not greater than 80% confluent: Check lot-specific characterization specification sheet for appropriate seeding density. Note: Some animal species create chains or islands of cells rather than being 100% confluent.
Please see the following causes and recommendations:
-Not enough time for cells to attach: Wait before overlaying with Geltrex Matrix to see if attachment increases; compare cultures to pictures on the lot-specific characterization specification sheet (human cells)
-Poor-quality substratum: Use Gibco Collagen I-Coated Plates
-Hepatocyte lot not characterized as plateable: Check lot specifications to ensure it is qualified for plating; review thawing, plating, and counting protocols
Please see the following causes and recommendations:
-Improper thawing technique: Review thawing, plating and counting protocols; thaw cells <2 mins at 37 degrees C
-Sub-optimal thawing medium: Use HTM Medium during thawing to remove cryoprotectant
-Incorrect centrifugation speed: Check thawing protocol for proper centrifugation speed and time (varies by species; human is 100 x g for 10 min at RT)
-Rough handling of hepatocytes during counting: Mix slowly, use wide-bore pipette tips; ensure a homogenous cell mixture prior to counting
-Improper counting technique: Count cells on 2 of the 4 grid lines; do not let cells sit in trypan blue mixture for more than 1 min prior to loading
Please see the following causes and recommendations:
-Improper thawing technigue: Review thawing, plating and counting protocols; thaw cells less than 2 mins at 37 degrees C
-Sub-optimal thawing medium: Use HTM Medium during thawing to remove cryoprotectant
-Rough handling of hepatocytes during counting: Mix slowly, use wide-bore pipette tips; ensure a homogenous cell mixture prior to counting
-Improper counting technique: Count cells on 2 of the 4 grid lines; do not let cells sit in trypan blue mixture for more than 1 min prior to loading
-Cells left out too long: Plate cells immediately after counting
Unlike immortalized cell lines, hepatocytes are primary cells that cannot be cultured indefinitely. The use of thawed suspension hepatocytes should be limited to short-term experiments with a maximum of 4-6 hour incubations. Plateable hepatocytes, which attach to collagen-coated plasticware in culture media, are generally metabolically active for anywhere from 2-7 days depending on which assay they are qualified for use with.
If properly stored, cryopreserved hepatocytes can be maintained in the vapor phase of liquid nitrogen (-135 degrees C or below) for several years. This makes them ideal for a series of experiments conducted over many months.
Cryopreserved hepatocytes are shipped in the vapor phase of liquid nitrogen contained in a non-hazardous container called a dry liquid nitrogen (LN2) vapor shipper or dewar. The internal temperature of the dewar is maintained between -140 degrees C and -160 degrees C, generally for up to 7-10 days if unopened. Once hepatocytes are received and transferred to a long term LN2 dewar in the laboratory, dewers are returned to the address listed on the pre-paid return shipping label for reuse. Upon receipt of a shipment of cryopreserved hepatocytes, carefully and quickly transfer the vials to the vapor phase of liquid nitrogen and keep at -135 degrees C or below until use. Any increase in the temperature of the cryovials before an experiment threatens the viability, functionality, and activity of the hepatocytes.