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Q: What media do you recommend for culturing these cells?

We recommend culturing HCECs in keratinocyte serum-free medium (KSFM), as the cells are manufactured and quality-controlled using this medium. However, if a defined growth system is preferred, we recommend defined keratinocyte serum-free medium (Defined Keratinocyte-SFM). Note that a Coating Matrix Kit is required for culturing HCECs in Defined Keratinocyte-SFM. In-house testing has shown comparable HCEC growth and morphology in both KSFM and Defined Keratinocyte-SFM.

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Q: Do HCECs require feeder cells or matrix coatings for proliferation?

A:  Feeder cells are not needed for culturing HCECs using KSFM or Defined Keratinocyte-SFM. However, HCECs cultured in Defined Keratinocyte-SFM require the use of a Coating Matrix Kit (recombinant type I collagen) for efficient attachment and growth.

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Q: What is the expected grow rate?

A:  When cultured under recommended conditions, HCECs grown in KSFM typically achieve growth rates of ~0.75–1.0 population doublings per day. Similar growth rates are seen using Defined Keratinocyte-SFM.

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Q: How long will HCEC cultures take to reach confluency?

A:  HCECs seeded at 5000 cells/cm2 typically reach ~90% confluency in 5–7 days when cultured as recommended.

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Q: What markers do they express?

A:  Each lot of HCECs is tested for positive expression of the widely accepted corneal epithelial markers p63α and cytokeratin 15 (CK15).

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Q: Where are HCECs isolated from?

A:  HCECs are isolated from normal human corneal-scleral tissue. Trimmed limbal regions are enzymatically digested, and released epithelial cells are expanded. HCECs are cryopreserved using a controlled-rate freezer at the end of the secondary culture (p1) in a cryopreservation reagent containing 10% dimethyl sulfoxide (DMSO).

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Q: How many times can I passage HCECs?

A:  HCECs are performance-tested and guaranteed to reach ≥12 population doublings after thaw. Generally, this requires 3–4 passages when HCECs are seeded at 5000 cells/cm2.

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Q: Are HCECs compatible with BacMam exogenous-gene delivery technology?

A:  In-house evaluation of BacMam (recombinant baculovirus-encoding mammalian-expression cassette) delivery technology has shown highly efficient expression in HCECs. BacMam 2.0 reagents, including CellLight®  fluorescent protein–signal peptide fusions and other BacMam-based biosensor products, typically yield transduction efficiencies of >70% under optimized conditions.

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Q: What type of cell morphology should I expect?

A:  HCECs display the typical cobblestone morphology observed with many types of epithelial cells grown in monolayer culture. Refer to the image on the HCEC product sheet for a representative phase-contrast image of cells approaching ~90% confluence. When cells are continually passaged, it is normal to see changes in morphology, including noticeable increases in average cell size. Cells may also appear more flattened and skirted. A reduction in the mitotic index or growth rate is usually observed in higher-passage cultures.

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