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On average, we have observed greater than 4-fold expansion per passage, but this can vary by cell line. Some cell lines can reach up to 10X expansion.

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Subculturing, also referred to as passaging, is the removal of the medium and transfer of cells from a previous culture into fresh growth medium, a procedure that enables the further propagation of the cell line or cell strain. Read more about cell growth and subculturing here (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/maintaining-cultured-cells.html.

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There are two basic systems for growing cells in culture, as monolayers on an artificial substrate (i.e., adherent culture) or free-floating in the culture medium (suspension culture). The majority of the cells derived from vertebrates, with the exception of hematopoietic cell lines and a few others, are anchorage-dependent and have to be cultured on a suitable substrate that is specifically treated to allow cell adhesion and spreading (i.e., tissue-culture treated). However, many cell lines can also be adapted for suspension culture. Similarly, most of the commercially available insect cell lines grow well in monolayer or suspension culture.

Cells that are cultured in suspension can be maintained in culture flasks that are not tissue-culture treated, but as the culture volume to surface area is increased beyond which adequate gas exchange is hindered (usually 0.2-0.5 mL/cm²), the medium requires agitation. This agitation is usually achieved with a magnetic stirrer or rotating spinner flasks.
Find a comparison chart between the two here (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-lines/adherent-vs-suspension-culture.html).

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Our Cell Culture Freezing Media is composed of DMEM, FBS, calf serum, and 10% DMSO. This is useful for many mammalian cells for freezing and is the same percentage of DMSO used in most methods. This product will work fine for most adherent cell lines grown with serum. A good general protocol for freezing cells can be found on our website by searching "Freezing cells protocol" from the home page.

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Insect cells are much more fragile than a lot of mammalian cell lines. They suffer much more damage than mammalian cells from overgrowth and over-splitting. Never let cells go above 8 x 10E6 cells/mL or grow at densities less than 0.5 x 10E6 cells/mL in suspension. Insect cells require a little more osmotic pressure than mammalian cells (340 µOsM). Insect cells use a lot of O2, especially during protein expression. Insect cell culture media is more acidic than mammalian media (pH 6.0-6.4). The insect cell culture media is phosphate buffer based. Therefore, no CO² is needed to maintain the pH.

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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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Yes, exosomes isolated with different surface markers can be distinctive in their protein profile. This has been demonstrated by Tauro et al. (http://www.ncbi.nlm.nih.gov/pubmed/23230278), who isolated two distinctive populations of exosomes based on surface markers EpCam or A33 from conditioned cell culture medium from a human carcinoma cell line. This proteomics study indicated that these two populations of exosomes are unique.

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The serum concentration will vary with the cell line and basal medium used. Please go here to see our recommended sera supplementations for tested cell lines (https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/classical-media/advanced-d-mem-and-mem/recommended-sera-supplementations.html).

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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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Most cell lines that are cultured using classic synthetic cell culture media, including MEM, DMEM, RPMI 1640, and DMEM/F-12 can be cultured with Gibco HPLM. There might be some differences in growth rates for some cell lines.

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The standard glass Lab-Tek slide cell culture treatment consists of a proprietary, multi-stage high-purity water washing process. Unlike our plastic products for adherent cell culture (see Nunclon Delta), there is no need for further modification of the surface because glass is already hydrophilic and therefore already suitable for culture of many adherent cell lines.

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Most normal mammalian cell lines grow well at pH 7.4, and there is very little variability among different cell strains. While most researchers usually use 5-7% CO² in air, 4-10% CO² is common for most cell culture experiments. Read more about buffering conditions here (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-environment/ph-CO²-levels.html).

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When cells meant to be grown in suspension are grown in static culture, they may form clumps. These clumps will severely limit transfection efficiency and protein expression. It is suggested that FreeStyle 293 cells in FreeStyle media and CHO-S cells in CD-CHO or CHO-SFM are grown in agitated suspension to reduce the appearance of clumps. However, if clumps do form, you can try the following protocol to select for cells that don't form clumps:

- Transfer cells into an appropriate size centrifuge tube that will hold the entire cell suspension.
- Allow cells to sit undisturbed for about 5 minutes. The time can vary depending on the specific cell line. Larger cell clumps will settle to the bottom of the tube.
- Collect cells from the upper portion of the tube to passage into a new flask.

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Diploid Growth Serum-Reduced Medium has been evaluated with fibroblast cell lines, including MRC-5, WI-38, IMR-90, BS-2, and CEF cells.

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