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Usually the best way to find out is to go to the source you obtained your cells from. For example, ATCC will have serum requirements for the cells they sell. Most insect cell lines and embryonic stem cell lines require heat-inactivated serum.

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Yes. We have evaluated over 10 different induced PSC (iPSC) and embryonic stem cell (ESC) lines, all of which were demonstrated to be compatible with StemScalePSC Suspension Medium.

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There have been multiple pluripotent stem cell (PSC) lines tested with the Gibco Essential 8 Medium System (https://www.ncbi.nlm.nih.gov/pubmed/?term=21478862).

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Cells isolated directly from tissue are primary cells. Once a confluent monolayer or a dense cell suspension is formed, the first subculture results in the formation of a cell line (Freshney, 1987, Culture of Animal Cells. A Manual of Basic Technique). This cell line has a finite lifespan (the Hayflick Limit), during which cells with the highest growth capacity will predominate, resulting in a degree of genotypic and phenotypic uniformity in the population. As the culture ages, it may go through a period of massive cell death (crisis), leaving transformed cells that will repopulate the flask. These cells are now a transformed (immortalized) cell line.

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When cells are isolated from a tissue to form a primary culture, assuming that the cells proliferate in vitro, a confluent monolayer or a dense cell suspension is formed. According to the traditional definition, the first harvesting and subculture of this cell population results in the formation of a cell line [Freshney, R.I. (1987). Culture of Animal Cells. A Manual of Basic Technique. (New York, Alan R. Liss, Inc.)]. This type of cell line has a finite lifespan, during which cells with the highest growth capacity will predominate, resulting in a degree of genotypic and phenotypic uniformity in the population.

Using this nomenclature system, a continuous cell line is a population of cells that has undergone a genetic transformation, resulting in indefinite growth potential. Continuous cell lines are usually aneuploid. In practice, continuous cell lines can be cultured through a very high number of subcultures, although some further genotypic, and therefore phenotypic, changes may occur at very high passage numbers. Immortalization can occur spontaneously or may be virally- or chemically- induced. Keep in mind that the working definitions of these terms can vary between research groups. Many researchers do not use the term “cell line” to refer to any population unless it has undergone a genetic transformation.

A cell strain is a subpopulation of a cell line that has been positively selected from the culture, by cloning or some other method. A cell strain will often have undergone additional genetic changes since the initiation of the parent line. Individual cell strains may, for example, have become more or less tumorigenic than the established line, or they may be designated as a separate strain following transfection procedures. The term cell type refers to all cells with a common phenotype, e.g., keratinocyte, melanocyte. Therefore keratinocytes isolated from a number of different donors are all the same cell type.

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Here is a list of cell lines that have been tested to work with the SuperScript IV CellsDirect cDNA Synthesis Kit:

Cell line; Culture properties; Organism; Tissue
- HeLa; adherent; Homo sapiens; cervical adenocarcinoma
- HeLa S3; suspension; Homo sapiens; cervical adenocarcinoma
- Raji; suspension; Homo sapiens; B lymphocyte
- NIH/3T3; adherent; Mus Musculus (mouse); embryonic fibroblast
- HEK-293; adherent; Homo sapiens; kidney
- Jurkat; suspension; Homo sapiens; acute T cell leukemia
- Daudi; suspension; Homo sapiens; Burkitt's lymphoma
- K562; suspension; Homo sapiens; bone marrow
- iPSC; adherent; Homo sapiens; stem cells
- Balb/3T3; adherent; Mus Musculus (mouse); embryonic fibroblast

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Mammalian cells are cryopreserved to avoid loss by contamination, to minimize genetic change in continuous cell lines, and to avoid aging and transformation in finite cell lines. Before cryopreservation, cells should be characterized and checked for contamination.

There are several common media used to freeze cells. For serum-containing medium, the constituents may be as follows:
1) Complete medium containing 10% DMSO (dimethylsulfoxide)
2) 50% cell-conditioned medium with 50% fresh medium with 10% DMSO

Cryopreservation media generally consists of a base medium, cryopreservative, and a protein source. The cryopreservative and protein protect the cells from the stress of the freeze-thaw process. A serum-free medium has generally low or no protein, but one can still use it as a base for a cryopreservative medium in the following formulations:

1) 50% cell-conditioned serum free medium and 50% fresh serum-free medium containing 7.5% DMSO
2) Fresh serum-free medium containing 7.5% DMSO and 10% cell culture grade BSA

Protocol for Suspension Cultures:
1. Count the number of viable cells to be cryopreserved. Cells should be in log phase.
2. Centrifuge the cells at ~200 to 400 x g for 5 min to pellet cells.
3. Using a pipette, remove the supernatant down to the smallest volume without disturbing the cells.
4. Resuspend cells in freezing medium to a concentration of 1 x 10E7 to 5 x 10E7cells/ml for serum-containing medium, or 0.5 x 10E7to 1 x 10E7 cells/ml for serum-free medium.
5. Aliquot into cryogenic storage vials. Place vials on wet ice or in a 4°C refrigerator, and start the freezing procedure within 5 minutes.
6. Cells are frozen slowly at 1°C /min. This can be done by programmable coolers or by placing vials in an insulated box placed in a -70°C to -90°C freezer, then transferring to liquid nitrogen storage.

Protocol for Adherent Cultures:
1. Detach cells from the substrate with dissociation agents. Detach as gently as possible to minimize damage to the cells.
2. Resuspend the detached cells in a complete growth medium and establish the viable cell count.
3. Centrifuge at ~200 x g for 5 min to pellet cells.
4. Using a pipette, withdraw the supernatant down to the smallest volume without disturbing the cells.
5. Resuspend cells in freezing medium to a concentration of 5 x 10E6 to 1 x 10E7 cells/ml. Aliquot into cryogenic storage vials.
6. Place vials on wet ice or in a 4°C refrigerator, and start the freezing procedure within 5 min.
7. Cells are frozen slowly at 1°C /min. This can be done by programmable coolers or by placing vials in an insulated box placed in a -70°C to -90°C freezer, then transferring to liquid nitrogen storage.
Reference: Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 220, Alan R. Liss,Inc., New York.

Thawing of Cryopreserved Cells:
Cryopreserved cells are fragile and require gentle handling. Thaw cells quickly and plate directly into complete growth medium. If cells are particularly sensitive to cryopreservation (DMSO or glycerol), they are centrifuged to remove cryopreservative and then plated into growth medium. The following are suggested procedures for thawing cryopreserved cells:

- Centrifugation Method: Remove cells from storage and thaw quickly in a 37°C water bath. Place 1 or 2 ml of frozen cells in ~25 ml of complete growth medium. Mix very gently. Centrifuge cells at ~80 x g for 2 to 3 min. Discard supernatant. Gently Resuspend cells in complete growth medium and perform a viable cell count. Plate the cells. Cell innoculum should be at least 3 x 10E5 cells/ml.
- Direct Plating Method: Remove cells from storage and thaw quickly in a 37°C water bath. Plate cells directly with complete growth media.

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The key applications include the following:

- Collagen I induces microvascular endothelial cells to adopt a spindle-shaped morphology, in vitro, and to align into solid cord-like assemblies. Vascular endothelial cells can also form vessel-like, tubular structures when cultured on collagen I. Collagen I can be used for in vitro angiogenesis assays.
- Breast cancer stem cells can undergo differentiation when cultured on collagen I.
- Collagen I has been used for the culture of primary colon carcinoma cell lines; mouse liver progenitor cells have been cultured in 3D collagen I and rat pancreatic islets.
- Collagen I can act as a barrier in cell invasion assays, and has been used to study cell adhesion.

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Yes, the TrypLE products contain rProtease, a non-animal, trypsin-like enzyme used for the dissociation of attachment dependent cell lines. TrypLE enzyme has demonstrated the ability to dissociate cells cultured both in serum-free and serum-supplemented systems. The catalog numbers for TrypLE Select are 12563-011 (100 mL) and 12563-029 (500 mL). Some example catalog numbers for TrypLE Express include 12604-013 without Phenol red (100 mL) and 12605-010 with Phenol red (100 mL). Both formulations are also available in additional sizes. All these products are animal origin-free.

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We recommend using TrypLE Express Enzyme or TrypLE Select Enzyme for passaging mesenchymal stem cells (MSCs). The usage of trypsin will result in lower cell viability upon passage. TrypLE Express and TrypLE Select are defined, animal origin-free, and stable at both room and refrigerated temperatures.

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PSC Definitive Endoderm Induction Kit consists of two animal origin-free media that enable efficient induction of human pluripotent stem cells (PSCs) to definitive endoderm.

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TrypLE reagent is free of animal- and human-derived components, producing an exceptionally pure reagent that is gentle on cells. Inactivation with trypsin inhibitors is not required. TrypLE reagent is ideal for dissociating a variety of attachment-dependent mammalian cell lines both in serum and in serum-free conditions, and can be directly substituted for trypsin in your current protocol. TrypLE reagent is room-temperature stable and ready to use when you need it. TrypLE cell dissociation reagents remain stable for 24 months at room temperature, making storage and handling easier and more convenient.

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As a general guideline, we recommend using Gibco B-27 Plus Supplement to culture neural stem cells, hippocampal, and other CNS neurons.

- For neuroblastomas, or post-mitotic neurons from both PNS and CNS, Gibco N-2 Supplement can be used.
- For primary glial cells (astrocytes) or tumor cell lines of astrocytic phenotype (astrocytes and gliomas), or oligodendrocytes, Gibco G-5 Supplement can be used.

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All the above products contain rProtease, a non-animal, trypsin-like enzyme formulation used for the dissociation of attachment-dependent cell lines. This alternative for porcine trypsin is a recombinant enzyme derived from microbial fermentation.

The TrpLE Express, TrypLE Select, and CTS TrypLE Select products differ in their production and testing methods and in the documentation provided.

TrypLE Select is for the industrial market and has release-testing both for endotoxins and enzyme activity. This material also has a Drug Master File on record. It is manufactured in AOF-dedicated equipment and is intended for Research Use/Further Cell Culture Manufacturing.

TrypLE Express is manufactured without use of dedicated equipment and is intended for Research Use Only. Not for use in diagnostic procedures.

CTS TrypLE Select is manufactured under scalable cGMP conditions using dedicated equipment and is designed for clinical applications. This product has a Drug Master File on record and is intended for Research Use or Manufacturing of Cell, Gene, or Tissue-Based Products. Not intended for direct administration into humans or animals.

To read more about TrypLE and cell release times using this reagent, please go here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin/tryple-express.html#1).

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The following is a suggested general procedure to rapidly remove various cell lines from the substratum while maintaining cellular integrity. This procedure is not meant to be universally applicable for all cell lines. The optimal conditions and concentrations employed for individual systems should be determined empirically. Cell viability should be routinely monitored at the time of subculturing. Cell viability should be greater than 90%.

1. Remove and discard spent cell culture media.

2. Wash cells using a balanced salt solution without calcium and magnesium or wash with EDTA. Add wash solution to the side of the flask opposite the cells. Rinse the cell sheet by rocking the flask for 1 to 2 min and discard wash solution.

3. Add the dissociation solution of choice at 2 to 3 ml/25 cm2 to the side of the flask opposite the cells. Be certain that the dissociation solution covers the cell sheet. Incubate the flasks at 37°C. Rock the flasks gently. Generally, cells are dissociated in 5 to 15 min. The actual time needed to dissociate cells will vary according to cell line. Monitor the process carefully to avoid cell damage. In addition to rocking gently, flasks of cell lines that are characteristically difficult to remove from the substratum may be tapped to expedite removal.

4. When the cells are completely detached, stand the flask in the upright position to allow the cells to drain to the bottom of the flask. Add complete media to the flask. Disperse the cells by pipetting repeatedly over the surface of the monolayer. Count and subculture the cells.

Reference:
Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 117, Alan R. Liss, Inc., New York.

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