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View additional product information for CELLstart™ Substrate - FAQs (A1014201)
32 product FAQs found
用于MSC培养和自我更新的生长因子：LIF（PHC9484），bFGF（PHG0261），EGF（PHG0311），HGF（PHG0324），PDGF（PHG0043）和Wnt3。
用于MSC分化的生长因子或细胞因子：BMP-2（PHC7145）与TGF-β1（PHG9211）。
(Arthritis Res Ther 9:204 (2007)).
MSC可培养在含或不含血清的培养基条件下。标准的培养条件为含10% FBS的低葡糖DMEM。MSC也可培养于低减血清（2%）的Gibco MesenPRO RS培养基（货号12746-012），或无血清的Gibco StemPro MSC SFM XenoFree培养基（货号A10675）和Gibco StemPro MSC SFM（货号A10332）中。通常情况下，MSC在含氧量较低的条件下（2% O2）生长得较好（2% O2）。
可通过流式细胞术来鉴定人体MSC，这些细胞通常具有CD73+，CD90+，CD105+，CD11b-和CD45-的标志物属性（Blood 109:4245 (2007)）。人MSC表达Oct4，Sox2和Rex-1；这些mRNA均可通过RT-PCR技术进行鉴定（Arthritis Res Ther 9:204 (2007)）。
间充质干细胞（MSC）是最初从骨髓或脂肪组织中分离出来的多专能细胞，它们表现出进一步分化为骨，软骨和脂肪细胞的能力。在常规细胞培养条件下，从骨髓中分离出的MSC为纺锤形，并拥有贴附于未包被塑料培养皿上的独特能力（Arthritis Res Ther 9:204 (2007)）。
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。
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。
We recommend to thaw the supplement in the 37°C water bath, aliquot and freeze at -5°C to -20°C. Thaw each aliquot only one additional time. To make 100 ml complete medium, mix 90.8 ml DMEM/F-12 with GlutaMax matrix, 2 ml StemPRO hESC supplement, 7.2 ml 25% BSA solution, 80 ul bFGF (at 10 ug/ml), and 182 ul 2-mercaptoethanol (at 55mM). Store at 2°C-8°C until ready to use, but use the same day.
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No, we don't recommend StemPro hESC SFM for mouse cells because they showed poor growth and some differentiation. We recommend KnockOut DMEM and KnockOut SR for mouse ESC cultures (catalog numbers 10829-018 and 10828-028 respectively).
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Yes, derivations using StemPro hESC SFM have been carried out using either a basement membrane extract (like Geltrex growth factor) or feeder cells.
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StemPro hESC SFM has been tested with the following hESC: H1, H9, BG01, BG02, BG03, HUES6, HUES9, Cyth203, CyT49, BG01v, HES-2, HES-3, KhES-1, and TE06. The medium has also been used with the Rhesus monkey line R336.4. Some cells may require a short adaptation time but most do not.
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Yes, we recommend using Geltrex hESC-qualified Reduced Growth Factor basement membrane (catalog number A10480-02), CELLstart (catalog number A10142-01) or similar matrix.
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In addition to the StemPRO hESC SFM kit, you will also need the following to make a complete medium: full length recombinant human FGF (catalog # PHG0261) and 2-Mercaptoethanol (catalog # 21985-023).
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The StemPro hESC SFM kit (Cat. No. A1000701) contains:
- 500 mL DMEM/F-12 with GlutaMax (Cat. No. 10565-018)
- 10 mL StemPro hESC SFM Supplement 50X (Cat. No. A10006-01)
- 40 mL Bovine Serum Albumin 25% (BSA Solution) (Cat. No. A10008-01)
Note that the StemPRO hESC Supplement and BSA solution cannot be ordered individually.
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CELLstart substrate has also been tested with the following hESC: RCM-1, H9, BG01v, I3, I6. CELLstart substrate has been tested with the following other primary and stem cells: human foreskin fibroblasts, mouse fibroblasts, H-9 derived NSCs and bone marrow-derived MSCs.
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CELLstart substrate has been tested with Stempro hESC SFM, Stempro MSC SFM, Stempro NSC SFM, Essential 8, and Mouse embryonic fibroblast (MEF) -conditioned medium generated using Knockout Serum Replacement.
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Yes, it's normal to have changes in morphology when switching to a new cell culture system. Even with differences in morphology, pluripotency is still retained.
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In general, yes. However, this may be dependent on the hESC line and the split ratio (seeding density) may need to be optimized to achieve the desired proliferation rate.
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We recommend using dishes that are coated less than 48 hours before use. Depending on the sensitivity of your cells, you may be able to coat plates and store them for up to two weeks before use. If the coated plates are stored, they should be stored at 2C-8C, with parafilm tightly sealing the plates. Bring the plates to room temperature and aspirate liquid before use.
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Yes, the cells can be plated from a single cell suspension. Ensure that the medium is supplemented with ROCK inhibitor to maintain pluripotency of hESCs.
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We recommend a 1:50 dilution for hESC applications. However, you may optimize this dilution based on your hESC line if you desire. For dilution instructions for MSC and NSC applications, see the product manual for StemPro MSC SFM or StemPRO NSC SFM.
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Yes, the gelling is a normal characteristic of CELLstart substrate, and we have provided sufficient volume in the vial to accommodate this gelling. The gelling does not have an impact on performance of the remaining product. Use only the liquid, allowing the gelled material to remain in the vial.
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Yes, enzymes can be used. We recommended TrypLE Select. We do not recommend collagenase or dispase due to lot variability of these products, animal origin composition, and the damage they can do to the cells.
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Growth factors used for MSC maintenance and self-renewal: LIF, FGF-basic, EGF, HGF, PDGF, and Wnt3.
Growth factors or cytokines for MSC differentiation: BMP-2and TGF-β1.
(Arthritis Res Ther 9:204 (2007)
View our Gibco™ MSC research products.
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MSCs may be cultured in either serum-containing medium or serum-free medium. The standard culturing conditions are DMEM, low glucose with 10% FBS. MSCs can also be grown in reduced-serum (2%) Gibco MesenPRO RS Medium (Cat. No. 12746-012), or in serum-free Gibco StemPro MSC SFM XenoFree Medium (Cat. No. A10675) and Gibco StemPro MSC SFM (Cat. No. A10332). In general, MSCs grow better under hypoxic conditions (2% O2).
Human MSCs can be identified by flow cytometry, typically displaying CD73+, CD90+, CD105+, CD11b-, and CD45- marker characteristics (Blood 109:4245 (2007)). Human MSC expresses Oct4, Sox2, and Rex-1; these may be verified using RT-PCR (Arthritis Res Ther 9:204 (2007)).
Mesenchymal stem cells (MSCs) are multipotent cells isolated primarily from bone marrow or fat tissues that exhibit the ability to differentiate into bone, cartilage, and fat cells. Under normal cell culture conditions, MSCs isolated from bone marrow are spindle shaped with the unique ability to adhere to uncoated plastic culture dishes (Arthritis Res Ther 9:204 (2007)).
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.
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.
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