Geltrex™ hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix - FAQs

人体ES细胞一般可通过以下方法进行鉴定：经典型的形态（它们以致密的细胞团形式生长 生长为紧密聚集的小型细胞团，细胞小并具有高核质比）；表面标志物的表达；干细胞特异性基因表达的RT-PCR检测（如Oct3/4，Sox2和Nanog）；碱性磷酸酶染色和端粒酶活性检测。最为常用的人ES细胞特异性表面标志物包括发育阶段特异性的胚胎抗原SSEA-3和SSEA-4。其它的ES细胞特异性的其它表面抗原还包括TRA-1-60和TRA-1-81。（Science 282:1145 (1998)）。

人胚胎干细胞源自人类囊胚内细胞团, 是通过使用兔抗血清检测BeWO细胞（人滋养层细胞系）抗血清的通过免疫外科法手段来进行分离的（Science 282:1145 (1998)）。

胚胎干（ES）细胞是源自早期哺乳动物胚胎的细胞，能够在体外进行无限的未分化增殖，同时保留分化为各类成体组织（包括生殖细胞）的潜能。ES细胞的多能性通常在体外通过以下方法来证明：将ES细胞诱导分化为类拟胚体，并以谱系特异性的标志物检测三个体胚层（内胚层，中胚层和外胚层）中分化细胞的谱系特异性标志物分化情况，或将这些细胞注射到免疫缺陷型小鼠中，以检测畸胎瘤中产生的细胞类型。

是的，这是正常情况。我们在产品开发过程中也见到了类似情形。请在预包被处理后吸去Geltrex 溶液，再按常规操作加入细胞。我们使用即用型Geltrex 基质预先包被的成品平板来培养H9人体胚胎干细胞，在进执行预包被步骤时有过类似发现。

是的，请尝试使用不同稀释浓度的基质来包被平板。一些客户使用1:100的稀释浓度获得了更好的结果。最佳稀释浓度取决于所使用的细胞系。

Gibco Geltrex 基质是含有细胞粘附所需的胶原IV，层粘连蛋白及其他基质蛋白和生长因子的基底膜抽提物。AlgiMatrix 基质只含有不具细胞粘附促进能力的藻酸盐。藻酸盐海绵体中没有能与细胞相互作用的成份。

即用型成品Gibco Geltrex 基质仅适用于为细胞粘附提供包被，主要为ESC和iPSC的应用进行研发。由于蛋白浓度不够高，它不适于厚凝胶或3D培养的相关应用。

是的，这是正常情况。我们在产品开发过程中也见到了类似情形。请在预包被处理后吸去Gibco Geltrex 基质溶液，再按常规操作加入细胞。我们在使用即用型成品Geltrex 基质预先包被的平板来培养H9人体胚胎干细胞时，在进行预包被步骤时也有过类似发现。

如果您希望从基质中取出细胞进行培养，我们推荐您首先润洗细胞，之后使用胰酶/EDTA，TrypLE 试剂，或使用低葡糖DMEM配制的3000U/mL胶原酶和1000U透明质酸酶溶液解离细胞。如果您希望对从基质中取出的细胞进行细胞膜表面标志物的染色，我们推荐您以机械方法取出细胞，如剧烈冲洗或仔细刮下。

请注意，如果细胞从基质中取出后培养于2D条件下，那么细胞的表型可能发生改变。

是的，这些产品就是在设计中就是配合使用的。

Gibco Geltrex 基质是减生长因子（reduced growth factor，RGF）基底膜抽提物（BME）的可溶性配方产品，是从鼠类Engelbreth-Holm-Swarm肿瘤中纯化获得的。在历史上这些制备产物中含有并不会影响产品效价的乳酸脱氢酶增高病毒（LDEV），但在某些如异种移植及其他临床性研究一类的应用中可能会引发顾虑。当前我们已通过全新的生产方法去除了这种些病毒。这种些不含LDEV的试材材料经我们使用PCR方法测试确定已不含LDEV——我们使用PCR方法确认了这些产品中不含病毒。

Gibco Geltrex 基质与胶原（大鼠尾与牛）可作为包被溶液或3D凝胶基质来使用。CELLstart 底物作为一种不含异外源性物质的包被基质，专为ESC的相关应用而研发（作为动物源性的Gibco Geltrex 基质或动物源性的Matrigel 基质的一种替代品底物）。AlgiMatrix 基质是为一种3D支架类的基质，它不支持并不为细胞提供粘附的基础，但而是为细胞球生长提供一个良好的生长环境，后续应用中也可方便易于细胞球的收取操作用于后续应用。

No, the Ready-To-Use Geltrex matrix was developed for use with the StemPro hESC SFM System. It is not recommended for other PSC media systems.

For other PSC media systems, we recommend the Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix (Cat. Nos. A1413301, A1413302).

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Geltrex hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix (Cat. No. A1569601) is a pre-diluted Geltrex product ready to use for coating culture vessels. There is no need to dilute the solution further. The product has also been QC tested using hESCs to ensure that every lot will maintain the attached pluripotency of these cells. The full product description can be found here:

Geltrex hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix (Cat. No. A1569601)

Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix (Cat. Nos. A1413301, A1413302) is a "concentrated" version of the Geltrex hESC-Qualified, Ready-To-Use above. You can pre-dilute this concentrated solution to pre-coat the culture vessel to grow hESCs at your desired usage concentration. This Geltrex matrix has also been QC tested using hESCs to ensure every lot will maintain the attached pluripotency of these cells. The full product description can be found here:

Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix (Cat. Nos. A1413301, A1413302)

Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Cat. Nos. A1413201, A1413202) is the same as the product above. However, it has not been tested to verify it will support hESC growth and expansion. This is a "general" version and not specific to a cell type or line. A full product description can be found here:

Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Cat. Nos. A1413201, A1413202)

Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Cat. Nos. A1413201, A1413202)

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

The Gentamycin sulfate concentration in Geltrex hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix (Cat. No. A1569601) is 10 µg/mL.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

For stem cells, a 1:100 dilution is the recommended starting dilution for Geltrex hESC, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix (Cat. No. A1569601). Other (lower) dilutions may be necessary for other primary cell types and should be determined empirically.

Geltrex hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix is in principle a diluted version of the frozen Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix.

Please take a look at page 2 under section “Recover frozen PSCs in complete StemFlex Medium” in the manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0016431_StemFlexMediumKit_UG.pdf) for the volume of Geltrex to use to cover a 6-well, 12-well, or 24-well plate.

Basically,
One well of a 24-well plate has a surface area of 2 cm² and you need 300 µL of diluted Geltrex per well.
One well of a 48-well plate has a surface area of 1 cm² and you need 150 µL of diluted Geltrex per well.
One well of a 96-well plate has a surface area of 0.32 cm² and you need 48 µL (˜50 µL) of diluted Geltrex per well.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

We do not have comparison data for the two Geltrex products. We recommend using the Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix for hepatocytes. We have not performed a significant amount of testing using the Geltrex hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix on these cells.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Human ES cells are generally characterized by their typical morphology (they grow as tightly packed clusters of small cells with high ratio of nucleus to cytoplasm); surface marker expression; RT-PCR detection of stem cell-specific gene expression (such as Oct3/4, Sox2, and Nanog); alkaline phosphatase staining, and telomerase activity assay. The most commonly used ES specific surface markers include stage-specific embryonic antigens SSEA-3 and SSEA-4 for human ES cells. Other ES-specific surface antigens also include TRA-1-60 and TRA-1-81. (Science 282:1145 (1998).

Human ES cells are derived from human blastocyst inner cell masses, isolated by immunosurgery with rabbit antiserum to BeWO cells (a human trophoblast cell line) (Science 282:1145 (1998)).

Embryonic stem (ES) cells are derived from the early mammalian embryo and are capable of unlimited, undifferentiated proliferation in vitro while maintaining their potential to differentiate into a wide of range of adult tissues including germ cells. The pluripotency of the ES cells is normally demonstrated in vitro by inducing ES cells to differentiate into embryoid bodies and checking lineage-specific markers for differentiated cells in three body layers (endo, meso, and ectoderm), or injecting them into immunodeficient mice and determining the cell types produced in the teratomas.

Yes, this is normal. It has been observed in product development. Remove the Geltrex solution after pre-coating and add cells as normal. H9 human embryonic stem cells have been cultured on dishes pre-coated with Ready-to-Use Geltrex Matrix with this observation in the pre-coating procedure.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Yes, try a variety of dilutions to coat the plates. Some customers use a 1:100 dilution and have better results. The optimal dilution is cell-line dependent.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Geltrex Matrix is a basement membrane extract that contains collagen IV, laminin, and other matrix proteins and growth factors that are required for cell adhesion. AlgiMatrix Matrix contains only alginate which does not facilitate cell adhesion. There is nothing in the alginate sponges for the cells to interact with.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Ready-to-Use Gibco Geltrex Matrix provides only a coating for cell attachment and was developed mainly for ESC and iPSC use. Because the protein concentration is not high enough, it should not be used for thick gel or 3D applications.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Yes, this is normal. It has been observed in product development. Remove the Gibco Geltrex Matrix solution after pre-coating and add cells as normal. H9 human embryonic stem cells have been cultured on dishes pre-coated with Ready-to-Use Gibco Geltrex Matrix with this observation in the pre-coating procedure.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

If you would like to culture the cells once recovered from the matrix, we recommend washing the cells first, then using trypsin/EDTA, TrypLE reagent, or a solution of 3,000 U/mL collagenase and 1,000 U hyaluronidase in low-glucose DMEM. If you would like to monitor cell surface markers, we recommend removal of the cells by mechanical means, such as a vigorous wash or a delicate scrape.

Note that the phenotype of the cells may change if they are cultured in 2D after their removal from the matrix.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Yes, these products were designed to work together.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Gibco Geltrex Matrix is a soluble form of reduced growth factor (RGF) basement membrane extract (BME) purified from murine Engelbreth-Holm-Swarm tumor. Historically these preparations have contained the lactate dehydrogenase-elevating virus (LDEV), which does not impact the performance of the material but may be of concern in certain applications such as xenografts or other clinically focused research. New manufacturing methods are now available that allow for the removal of this virus. The resulting LDEV-free material is tested using PCR methods to confirm that the material is virus-free.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Gibco Geltrex Matrix and collagen (rat tail and bovine) can be used as either a coating solution or a 3D gel matrix. CELLstart Substrate was developed to be used as a xeno-free coating matrix for only ESC applications (as a substitute for Gibco Geltrex Matrix or Matrigel Matrix, which are of animal origin). AlgiMatrix Matrix is a 3D scaffold-type matrix that does not support cell attachment but does provide a good environment for growing spheroids that can be easily harvested for downstream applications.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.