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Method Article
This protocol outlines the culture of freshly obtained (passage 0) keratinocytes for use in live cell imaging.
Tracking stem cells and committed progenitor behavior at the single-cell level in human skin has been challenging both in vivo and in vitro. Live-cell imaging has allowed for significant advances in the ability to identify differences between keratinocyte stem cells and committed progenitor behavior. Live-cell imaging is an evolving and somewhat challenging method to study keratinocyte behavior in vitro. This protocol has been developed to culture keratinocytes at low seeding density, enabling relatively long-term time-lapse photography and monitoring of individual cell behavior. Passage 0 keratinocytes are grown at clonal density, and time-lapse photography allows documentation of individual cell divisions and the time of their occurrence. For maximum biological relevance, freshly isolated human keratinocytes are placed in vitro. This approach focuses on proliferation. However, this protocol can be adapted for use in other live cell imaging applications measuring individual cell behavior, such as measuring cell migration, wound healing, and motility.
The goal of this protocol is to provide the ability to track individual keratinocytes in culture over a relatively long period (several weeks) enabled by low seeding densities and time-lapse photography. Live cell imaging at low density affords investigators the opportunity to visualize cell behavior in vitro at a single cell level. While not possible in conventional cell culture, live cell imaging makes it possible to develop lineage trees in real-time, without the use of labeling, from which one can ascertain information regarding division kinetics, such as cell cycle duration and the proportion of proliferation and differentiation divisions in a colony. It also allows for the study of cell migration and motility. It can be technically challenging, with aspects that need optimization, depending on the cell type and on the data that is to be captured. Other labs have utilized neonatal keratinocytes and/or passaged keratinocytes that are easier to grow1.
However, cell lines that undergo repeated passaging are significantly different in behavior and morphology from their in vivo state2. For the greatest biological relevance in order to obtain the closest to in vivo behavior, passage 0 cells are utilized. In keratinocyte populations, it is possible to distinguish between the stem cell and committed progenitor without sorting or labels because there are distinct differences in division behavior that can be observed via live cell imaging3.
We utilize live cell imaging to study keratinocyte proliferation kinetics. A similar approach has been used to study the motility of cutaneous melanoma cells cocultured with keratinocytes4, adherent cell migration of scratch assays5, and how ROCK inhibitors affect keratinocyte proliferation6. This protocol is specifically designed for culturing cells to facilitate lineage tracing by live cell imaging, although it may be adapted for other purposes.
This protocol outlines the isolation of passage 0 keratinocytes for the purpose of live cell imaging, discusses complications that may arise, and provides considerations that may require alterations to the protocol (Figure 1).
Approval from the institution's Committee on Human Research is required for the use of human tissue. Human samples for research are often obtained from commercial sources (for example, ATCC, Lifeline cell technology, and Zen-Bio). Our studies use discarded tissue at the time of surgeries or skin biopsy samples taken specifically for study purposes. These situations require different approvals and consenting procedures. All tissues are obtained after approval from the UCSF Committee on Human Research, utilizing written informed consent and following the tenets of the Declaration of Helsinki.
1. Separation of epidermis from the dermis
2. Isolation of keratinocytes
Given the multiple variables that may be manipulated in this method that ultimately result in a variety of outcomes, here we outline common missteps and their resultant outcomes, as well as provide exemplar outcomes and the circumstances behind them.
As with any cell culture technique, contamination is a possibility (Supplementary Video 1). Along with careful sterile techniques, consider using antibiotics in media, especially if culturing for extended periods of time. Frequent...
Here, we have detailed a method for the primary culture of human keratinocytes for the purpose of live cell imaging. This method adapts existing cell culture techniques using low-density plating and long-term culture to allow for successful live cell imaging studies. The preferred method for dissociation of this cell type involves a two-step enzymatic digestion, which has been shown to result in keratinocytes, of which 3%-4% are capable of becoming colony-forming units14. Adult and aged kerat...
The authors have nothing to disclose.
Special thanks to T. Richard Parenteau, MD/PhD, and Alexandra Charruyer, PharmD/PhD, for teaching me how to culture keratinocytes. Thanks to Merisa Piper MD for providing us with samples to isolate keratinocytes from. Final thanks to Michael Rosenblum MD for allowing us access to his live cell imaging system to complete the experiments.
Name | Company | Catalog Number | Comments |
.05% Trypsin-EDTA (1X), 100 mL | Gibco | 25300-054 | |
100 um cell filter | Corning | 352360 | |
15 cc Falcon | Corning | 352096 | |
50 cc conical Falcon | Corning | 352070 | |
6 well microplate | Corning | 3516 | |
70% reagent alcohol, 4 L | VWR Chemicals | BDH1164-4LP | Can alternatively dilute your own |
Amphotericin B, 50 ml | Corning | 30-003-CF | Dilute to 5X (50 ug/mL) |
Dipase, 100 ml | Corning | 354235 | Dilute 1:1 with HBSS, add 1x gentamycin, filter sterilize |
Epilife, 50 mL | Gibco | MEP1500CA | Add HKGS, consider antibiotics |
Fetal Bovine Serum Value Heat Inactivated FBS, 500 ml | Gibco | A52568-01 | FBS |
Forceps | |||
Gentamicin, 10 ml | Gibco | 15750-060 | Dilute to 50 ug/ml for 5X |
Hank's Balanced Salt Solution (1X), 500 ml | Gibco | 14170-112 | (HBSS) |
HBSS 5X PSAG | This is HBSS + 5X concentrations of Pen/Strep/Ampho/Gent | ||
Hibiclens, Gallon | Molnlycke | 57591 | Dilute to 10% using deionized H20 |
Human Keratinocyte Growth Serum, 5 mL | Gibco | S-001-5 | Added to epilife |
Penicillin/Streptomycin, 100 ml | Corning | 30-002-Cl | Dilute to 5X (comes in 100x stock) for 5X PSA - 1X for media changes |
Petri Dish 100 mm x 15 mm | Fisher Scientific | FB0875713 | |
Scalpel Blade NO 23 | VWR | 76457-480 | |
TrypLE | Gibco | 12604021 | A less caustic alternative to regular Trypsin |
Trypsin Neutralizing Solution | (TNS), this is HBSS with 10% FBS (some use less serum, 5%) |
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