CELLstart™ Substrate - FAQs

How do I prepare the StemPRO hESC SFM complete medium?

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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Can StemPRO hESC SFM be used to culture mouse ESCs?

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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Can StemPRO hESC SFM be used for new cell line derivations?

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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What cell lines has StemPRO hESC SFM been tested with?

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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Does StemPRO hESC SFM require the use of a basement membrane matrix?

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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What additional products do I need to make a complete StemPRO hESC SFM?

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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What are the components of the StemPro hESC SFM kit?

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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Which cell lines has CELLstart substrate been tested with?

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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What media has CELLstart substrate been tested with?

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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The morphology of my cells looks different when growing on CELLstart substrate. Is this normal?

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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Will my cell passaging ratio remain the same when culturing cells on CELLstart substrate?

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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Can I coat plates with CELLstart substrate and store them for later use?

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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Can hESCs be subcultured in single cell suspension when growing on CELLstart substrate-coated plates?

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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Can I dilute CELLstart substrate beyond the recommended 1:50 dilution?

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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Can I use the CELLstart substrate if there is gelling in the vial?

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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Can enzymes be used to passage cells cultured on CELLstart CTS substrate?

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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What kinds of growth factors are used in mesenchymal stem cell (MSC) research?

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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How can I culture mesenchymal stem cells (MSCs) in vitro?

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).

How do you characterize human mesenchymal stem cells (MSCs)?

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)).

What are mesenchymal stem cells?

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)).

How do I characterize human embryonic stem ( ES) cells?

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).

How are human embryonic stem (ES) cells derived?

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)).

What are ES cells?

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.

What are some general differences between Gibco Geltrex Matrix, collagen, AlgiMatrix 3D Culture System, and CTS Cellstart Substrate?

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.