Help & FAQs: Stem Cell Products

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Yes. The N-2 MAX Media Supplement may be stored frozen as 1 mL aliquots. The frozen aliquots should not undergo multiple freeze-thaws before use.

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The following tips/hints are useful for chondrogenic differentiation:a) The mesenchymal stem cells (MSCs) should not be from a late passage (passage 8 or less), b) if using the Human Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC006) or the StemXVivo® Chondrogenic Supplement (Catalog # CCM006), use the starting MSC cell number that is indicated in the protocol, c) Early during chondrogenic differentiation a pellet should form. As differentiation progresses, the pellet will grow and take up a ball-like appearance. d) The pellet should not attach to the tube, therefore care should be taken to not dislodge it while changing media.

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Coating glass cover slips with Cultrex Laminin I is quite common and has been published extensively.

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This kit is unlikely to work with human MSCs as these cells are at a different point in development and are expected to need different signals.

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MSCs grown in StemXVivo® MSC Expansion Media can be cryopreserved using appropriate cryopreservation media, such as StemXVivo® Serum-Free MSC Freezing Media (Catalog # CCM016) or equivalent.

Question 6

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Yes ,StemXVivo® Mesenchymal Stem Cell Expansion Media contains Phenol Red.

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The StemXVivo® Mesenchymal Stem Cell Expansion Media does not need addition of serum. Fetal bovine serum is included in the product, which is a complete medium and ready to use. The media may be supplemented with cytokine or growth factors for your desired cell culture application.

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No. The N-2 MAX Media Supplement does not contain any animal-derived components other than Human Transferrin. GMP N-2 MAX Media Supplement is an animal-free supplement (Catalog # AR016).

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Organoids or 3-D cultured cells may be harvested using the Cultrex® 3-D Culture Cell Harvesting Kit (Catalog # 3448-020-K)

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The cells can be cultured for approximately one week. After this period, loss of cell viability is observed.

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MSCs can be grown to 80-90% confluency and subsequently subcultured using the protocol provided in the product datasheet. Researchers should establish the number of passages that is acceptable for their work. MSCs are sensitive to passsages and, if subcultured too many times, may start losing their MSC characteristics. Our MSC Functional Identification Kits can be used for validation of MSC mulitpotency.

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Beating cells can be seen as early as Day 10-13 of differentiation. Beating then becomes more widespread throughout the following week.

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The Mouse Cortical Stem Cells are designated P1 cells as the supplied cells are passaged once. They can be expanded at least three more passages using the monolayer system or at least four more passages using the neurosphere system before their multipotency may be compromised.

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Cells need to be healthy and actively dividing in 2-D culture. Cells should be passaged two or three times after resuspension from cryopreservation, and they should never surpass 80% confluency during each passage. Cells should also be assessed for viability using trypan blue, and they should exhibit less than 5% staining.

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Yes, the components are a available individually. However, reagents will not be matched and they will not have undergone Quality Control testing together in the application for which the kit was designed. One additional concern is that once the proteins are reconstituted, they have a limited shelf-life that we can guarantee. Shelf-life is maximized by only packaging the quantity needed for a defined amount of media. Kits are designed so that all components are used at the same rate. If there is a problem, please contact Technical Service.

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The Irradiated Mouse Embryonic Fibroblasts will most likely not survive a second freeze-thaw. Freezing the cells after one use is not recommended.

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Yes, the StemXVivo® Human Adipogenic Supplement (Catalog # CCM011) , StemXVivo® Human Osteogenic Supplement (Catalog # CCM008), and StemXVivo® Human Chondrogenic Supplement (Catalog # CCM006) are the same as Part #'s 390415, 390416, and 390417, respectively, in the Human Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC006).

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We have not compared, in a side-by-side experiment, medium with and without ribonucleosides and deoxyribonucleosides for growing mesenchymal stem cells. There is literature to support the fact that using medium without ribonucleosides and deoxyribonucleosides is beneficial for growing mesenchymal stem cells.

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Yes. The crystals are a calcium-containing precipitate and may form, to a limited degree, in this media. A limited amount of crystals will not impact MSC expansion. Avoiding multiple freeze-thaw cycles of the medium is helpful in reducing the amount of crystals.

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Fico/Lite cell separation media should always be stored at refrigerated temperatures. Upon freezing any Fico/Lite product, gradients can form. Slowly thaw the solution and carefully mix it. If no precipitation is visible, the product should still be suitable for use.

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3-D cultures are in vitro cultures where immortalized cell lines, primary cell lines, stem cells, or tissue explants are placed within hydrogel matrices, such as Cultrex Basement Membrane Extract, that mimic in vivo cell environments and allow cells to proliferate in three dimensions.

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Cultrex Rat Collagen I is provided at 5 mg/mL and is not pepsin treated, so it has intact telopeptides and is highly viscous. Cultrex Rat Collagen I, Low Viscosity is provided at 3 mg/mL, and it is easier to pipet. Both are provided in acetic acid and require pH neutralization prior to use. Please consult the provided product data sheets for pH neutralization instructions.

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EPO is a potent cytokine used to stimulate differentiation of HSCs. EPO is needed for erythrocyte development. Media without EPO offers researchers the ability to expand and differentiate their HSCs and then to test supplemental drugs or compounds of interest.

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The two principal methods for performing 3-D culture are the top assay and embedded assay. For the top assay, cells are seeded on a thick gel of Cultrex Basement Membrane Extract (BME) or Extracellular Matrix Protein. A thin overlay of cell culture medium is then applied to the cells. For the embedded assay, cells are resuspended within a thick gel of Cultrex BME or ECM and the culture media is applied on top. The top assay is easier to setup, to control seeding densities, and to keep cells within one focal plane for analysis.

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The major variables associated with 3-D culture are cell type, cell seeding density, composition of hydrogel, thickness of hydrogel, stiffness of hydrogel, composition of cell culture medium, and time of culture.

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Laminin I is a major component of extracellular matrix. Cultrex Laminin I is purified from murine EHS sarcoma. It is composed of α1β1γ1 chains with a total MW of 800,000 Da. Cultrex Laminin I increases cell adhesion, migration, growth, differentiation, neurite outgrowth, protease production, and malignancy. The response is dependent on cell type.

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Environmental stewardship is important to R&D Systems and its employees. R&D Systems is ISO 14001:2015 Certified and as part of this we are continuously reviewing our sustainability practices and "green" options. First and foremost, the energy expended to ship back the styrofoam box is more detrimental to the environment than having the facility re-use or recycle it. Our stance is to encourage our customers to implement a recycling program locally. At R&D Systems we have chosen to reduce the use of styrofoam as much as possible by: doing extensive stability testing in order to determine which products can be shipped with minimal packaging at ambient temperatures, converting the use of non-recyclable packaging materials to recyclable plastics, cardboard, or biodegradable materials, and continuing to investigate alternatives to dry ice shipments, the use of re-usable containers, and gel packs that allow for smaller styrofoam containers. Employees were key to initiating a recycling program at our facilities. Internally, we recycle paper, plastic, cardboard, aluminum, and glass.

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While 2-D culture has been used for studying many aspects of cell function and behavior, the tissue-culture treated plastic environment is unlike anything found within living organisms. As a result, cells in 2-D culture exhibit altered morphology, function, proliferation, and gene expression when compared to their emanating tissues. By placing these cells in a 3-D environment, they assume biological and biochemical characteristics similar to what is observed in vivo.

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Only the source species for the protein is different. Both carrier-free proteins have been validated to be bioactive with the same assay protocols listed and recommended concentrations fall within the same range, although customers should determine their optimal concentrations for their particular cell type and application.

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The Methylcellulose Stock (Catalog # HSC001) contains methylcellulose in Iscove's Modified Dulbecco's Medium. The Base Media (Catalog # HSC002) contains methylcellulose in Iscove's Modified Dulbecco's Medium along with serum, albumin, L-glutamine, and 2-mercaptoethanol. The additional components of HSC002 are needed to sustain the viability and growth of HSCs. The advantage to using HSC002 is that R&D Systems has already evaluated the serum, albumin, L-glutamine, and 2-mercaptoethanol in-house to ensure lot-to-lot consistency, eliminating the need for the researcher to evaluate these components individually.

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The N-2 Plus Media Supplement (Catalog # AR003) contains Bovine Insulin whereas N-2 MAX Media Supplement (Catalog # AR009) contains Recombinant Human Insulin. All other media components and concentrations in N-2 Plus and N-2 MAX are the same. These two products perform comparably in side-by-side testing. Due to a limited supply of bovine insulin, the products are priced differently.

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The Complete Methylcellulose Media (Catalog # HSC003) contains four growth factors (rhSCF, rhGM-CSF, rhIL-3, and rhEPO) whereas the Enriched Media (Catalog # HSC005) contains six growth factors (rhSCF, rhGM-CSF, rhIL-3, rhEPO, rhG-CSF, and rhIL-6) for hematpoietic stem cell (HSC) differentiation.HSC003 is typically used with an unpurified heterogeneous population of cells. PBMCs may be mixed with HSCs and the included growth factors will stimulate the PBMCs to express other growth factors to promote differentiation of HSCs.HSC005 is typically used with purified HSCs and it includes growth factors to differntiate HSCs without assistance from growth factors expressed from PBMCs.

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Cultrex 3-D Rat Collagen I undergoes the same basic purification and efficacy tests as the standard collagen. However, it undergoes additional 3-D culture validation; it has been tested extensively for the ability to promote growth and differentiation of cell types, visualized by morphology in three dimensions in vitro.

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Mouse cortical Stem cells are deived by harvesting brain tissue from embryos of pregnant females.Cortical Stem cells from each litter is combined giving a mixture of male and female cells.

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The recommended working concentration for thin coating is 0.05-10 µg/cm2 . However, conditions must be optimized for each cell line or model. Cultrex Laminin I should be used at 6 mg/mL for 3-D culture applications.

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The following medium is used in-house for cryopreserving mouse small intestinal organoids: 90%FBS + 10% DMSO + Y-27632 (10uM). Organoids are placed in cryovials (0.5mL) and frozen at -80C for up to a week and then moved to liquid nitrogen for long-term storage.

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Within the organoid, spheroid, or 3-D culture, cells may be assessed for morphology, apical/basal polarity, protein localization, and relative proliferation. In addition, cells may be isolated from the 3-D culture and evaluated for levels of RNA and protein expression, as well as modifications to DNA.

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The switch to maintenance media can be made any time after beating is initiated or upon the completion of the reagents provided in the kit.

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Choice of matrix should correspond to the environment that you wish to recapitulate. Cultrex Basement Membrane Extract (BME) will recapitulate the basal lamina, which underlie most cells of epithelial or endothelial origin. Cultrex Collagen I is the major constituent of connective tissue, and is commonly inhabited by stationary cells, such as fibrocytes and adipose cells, as well as migrating cells, such as mast cells, macrophages, monocytes, lymphocytes, plasma cells, and eosinophils.

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Sodium pyruvate is added to many low glucose and high glucose DMEM formulations. Sodium pyruvate can be used by cells as a readily accessible carbon source for energy production and other critical metabolic pathways, bypassing the need to produce it biosynthetically from glucose or amino acids. Some cell lines require the addition of pyruvate to the culture media since they lack the ability to convert glucose or amino acids into pyruvate.

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The cells are differentiated to the definitive endoderm stage by around day 4 from differentiation initiation. A schematic of the differentiation progression can be viewed in this scientific poster: https://resources.rndsystems.com/images/site/rnd-systems-stem-cell-derived-hepatocyte-like-cells-isscr-2016.pdf

Question 44

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This kit is unlikely to work with human MSCs as these cells are at a different point in development and are expected to need different signals.

Question 45

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The Human/Mouse/Rat Neural Lineage Functional Identification Kit has been tested with embryonic neural progenitor cells. The kit should also work with adult neural progenitor cells, as adult cells have similar growth conditions.

Question 46

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It is likely that the antibodies included in the kit are cross-reactive to other primates. The supplements included in the kit are not intended to be species-specific. However, the kit has not been tested with primate mesenchymal stem cells

Question 47

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The kit may work with a starting population of neural progenitor cells, but we have not tested this. If starting with neural progenitor cells we recommend starting at Stage 5 of the Dopaminergic Neuron Differentiation Kit protocol. The starting cell density would also have to be optimized.

Question 48

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For adipogenic differentiation, the appearance of vacuoles in cells after 5-7 days is a sign of differentiation and can be monitored by microscopic examination of the cells. For osteogenic differentiation, the beginning of cell detachment after about 14 days is a sign of differentiation. Cell detachment should be monitored in this case. For chondrogenic differentiation, there isn't an exact marker to look for other than fixing and staining the frozen pellet between differentiation days 14 - 21. The exact choice of time may take some empirical testing.

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Beating cells can be seen as early as Day 10-13 of differentiation. Beating then becomes more widespread throughout the following week.

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Following differentiation NPCs can be passaged approximately five times. However, it is ideal to cryopreserve the NPCs following the 7-8 day differentiation and save the frozen cells for future experiments.

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Yes, the components are a available individually. However, reagents will not be matched and they will not have undergone Quality Control testing together in the application for which the kit was designed. One additional concern is that once the proteins are reconstituted, they have a limited shelf-life that we can guarantee. Shelf-life is maximized by only packaging the quantity needed for a defined amount of media. Kits are designed so that all components are used at the same rate. If there is a problem, please contact Technical Service.

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Yes, the StemXVivo® Human Adipogenic Supplement (Catalog # CCM011) , StemXVivo® Human Osteogenic Supplement (Catalog # CCM008), and StemXVivo® Human Chondrogenic Supplement (Catalog # CCM006) are the same as Part #'s 390415, 390416, and 390417, respectively, in the Human Mesenchymal Stem Cell Functional Identification Kit (Catalog # SC006).

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One recommendation is to maintain the NPCs at a cell density of about 50,000-100000 cells/cm2. The second is to allow the NPCs to go through 2-4 passages before differentiating into astrocytes and oligodendrocytes as older NPCs show better differentiation into these neurons.

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For re-plating the day 19 hepatocytes, we used plates coated with Ultimatrix (catalog # BME001) or other Cutltrex BME. The medium used was Differentiation Base media II + Differentiation Cocktail IV (as described in Catalog # SC033). The cell density ranged from 3 x104 to 2 x 105. Addition of dexamethasone (50 µM) improved albumin expression.

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The N-2 Plus Media Supplement (Catalog # AR003) contains Bovine Insulin whereas N-2 MAX Media Supplement (Catalog # AR009) contains Recombinant Human Insulin. All other media components and concentrations in N-2 Plus and N-2 MAX are the same. These two products perform comparably in side-by-side testing. Due to a limited supply of bovine insulin, the products are priced differently.

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Yes. During NPC maintenance, cells should be cultured on plates coated with Cultrex® RGF BME. Coating a plate with Cultrex Poly-L-Lysine (Catalog # 3438-200-01; 10 µg/mL) followed by Cultrex Laminin (Catalog # 3400-010-02; 20 µg/mL) is another option.

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We have been successful in re-plating the Day 19 hepatocytes cells and maintaining the differentiated hepatocytes for 10 additional days.

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In-house, we have been able to cryopreserve day 9 progenitors.

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We have not done any gene editing work in-house but citations for CRISPR hepatocytes are available. https://www.sciencedirect.com/science/article/pii/S2589555921001658

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We have not needed to adapt iPSCs prior to differentiation, however, if iPSCs have been grown with Matrigel it may be helpful to have 1-2 passages in Cultrex BME.

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The switch to maintenance media can be made any time after beating is initiated or upon the completion of the reagents provided in the kit.

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The Human Mesenchymal Stem Cell Multi-Color Flow Kit (catalog # FMC002) or Mouse Mesenchymal Stem Cell Multi-Color Flow Kit (catalog # FMC003) will not work for identificaiton of Rat Mesenchymal stem cells.

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