Abstract
Mouse epidermal keratinocytes have traditionally been difficult to grow in vitro. In this chapter, we present a method for isolating epidermal keratinocytes from a single, newborn mouse pup for long-term culture. The protocols we describe will be especially useful for the isolation and analysis of cells harvested from transgenic or knockout mice. We explain how to use a supplemented fibroblast-conditioned medium, along with mouse collagen IV-coated culture dishes, to establish and subculture these fastidious cells for multiple passages. We describe how to induce expression of markers of the late stages of epidermal differentiation in cultured cells and how to ship whole mouse skins for culture at a site removed from the mice, should it be required. This chapter also contains a method of cryopreservation that ensures high cell viability after periods of storage over liquid nitrogen. The techniques described here in detail should be of interest to investigators currently producing transgenic or null mice with epidermal defects.
Key Words
1 Introduction
The analysis of transgenic or knockout mice with epidermal defects can be enhanced by in vitro studies. The isolation of murine epidermal keratinocytes from a single, transgenic, or knockout mouse pup for long-term culture is an important tool in these investigations. To be successfully maintained and subcultured, mouse keratinocytes require supplemented fibroblast-conditioned medium, a low-calcium environment, and culture dishes coated with mouse collagen IV for attachment. In this chapter, we describe how to make the mouse fibroblast-conditioned medium that is necessary for mouse keratinocyte growth, as well as how to extract and culture cells from individual newborn mouse skins. We include tips for effective subculture with collagen-coated dishes and how to induce expression of markers of the late stages of epidermal differentiation. We also present a method for transporting mouse skins for culture at a site removed from the mice, as well as a protocol for cryogenic preservation of viable cultured keratinocyte stocks. The collection of procedures outlined in this chapter should be of great interest to biologists seeking to augment their investigations of epidermal differentiation through the production of mouse models.
2 Materials
2.1 Chelexed Fetal Bovine Serum (cFBS)
-
1.
Chelex 100 Resin (sodium, 200–400 dry mesh, 75 to 150-μm wet bead; Bio-Rad, Hercules, CA, cat. no. 142-2842); use 200 g Chelex 100 Resin/L of FBS (1).
-
2.
Whatman Grade No.1 filter paper (Whatman, UK, cat. no. 1093-111) and Buchner funnel (optional, see Note 1 ).
-
3.
Endotoxin-free Milli-Q dH2O (18 megohm-cm resistance, from Milli-Q Plus-UF Water System or similar endotoxin-free water from a type I water filtration system; Millipore, Bedford, MA). When chelexing 1 L of serum, use 1.30 L endotoxin-free Milli-Q dH2O for each rinse (see Subheading 3.1.1. ).
-
4.
Dulbecco’s phosphate-buffered saline (D-PBS; ref. 2): 0.2 g of KCl, 0.2 g KH2PO4, 8 g NaCl, 2.16 g Na2HPO4-7H2O; To make 1 L of 1X D-PBS, add all dry chemicals to 600 mL endotoxin-free Milli-Q dH2O; adjust to pH 7.4 and bring to 1-L final volume; sterile filter (0.22 μm) into sterile bottles in a laminar flow hood; store at 4°C. When chelexing 1 L serum, use 1.30 L d-PBS for each rinse (see Subheading 3.1.1. ).
-
5.
FBS: serum lots are tested for optimal growth and differentiation of human newborn foreskin keratinocytes and a rat epidermal keratinocyte cell line (current lot is “Premium,” nonheat-inactivated; Atlanta Biologicals, Norcross, GA; cat. no. S11150).
-
6.
500 mL of bottletop filters: 70-mm membrane diameter, pore size 0.45 μm for initial filtering and 0.22 μm for sterile filtering (Corning, Harrodsburg, KY, cat. nos. 430512 and 430521, respectively) of cFBS.
2.2 Primary Fibroblast Conditioned Medium (CM1)
-
1.
Use newborn (d 0–2) mice (see Note 2 ).
-
2.
1 M CaCl2 solution: dissolve CaCl2•2H2O (FW 147.02) in endotoxin-free Milli-Q dH2O; 0.22 μm sterile-filter.
-
3.
100×15-mm Bacteriological petri dish (BD Falcon Optilux™ or similar style; Becton Dickinson, Franklin Lakes, NJ; cat. no. 351001).
-
4.
Whatman Grade No.1 filter paper (see Subheading 2.1. , item 2): cut to fit 100-mm Petri dish; wrap in aluminum foil and autoclave to sterilize before use.
-
5.
70% EtOH.
-
6.
D-PBS (see Subheading 2.1. , item 4).
-
7.
D-PBS with added 2% antibiotic/antimycotic (v/v) (antibiotic/antimycotic containing 10,000 U/mL penicillin G sodium, 10,000 μg/mL streptomycin sulfate and 25 μg/mL Amphotericin B; Invitrogen/GIBCO, Carlsbad, CA; cat. no. 15140-022).
-
8.
High-calcium medium (HCM): 100 mL Eagle’s minimal essential medium (EMEM) without calcium chloride (with Earles’ balanced salt solution, non-essential amino acids and l-glutamine; Cambrex Biosciences, Walkersville, MD; cat. no. 06-174G); 8 mL FBS; 1 mL antibiotic/antimycotic; add 1 M CaCl2 solution to bring medium to 0.6 mM Ca2+ (see Note 3 ). Store HCM up to 1 mo at 4°C/darkness.
-
9.
Low-calcium medium (EMEM.06): 100 mL EMEM without calcium chloride; 8 mL cFBS; 1 mL antibiotic/antimycotic; add 1 M CaCl2 solution to bring medium to 0.06 mM Ca2+ (see Note 4 ). Store up to 1 mo at 4°C/darkness.
-
10.
Collagenase solution: 0.175 g collagenase (crude, type I, for tissue culture; Invitrogen/GIBCO; cat. no.17100-017); 50 mL Medium 199 (Invitrogen/GIBCO, cat. no. 12340030). Make fresh as needed and warm to 37°C just before use.
-
11.
0.25% Trypsin (Invitrogen/GIBCO, cat. no. 15050-057): thaw a fresh aliquot the day of use and keep at 4°C until needed.
2.3 Coating Culture Dishes With Mouse Collagen Type IV
-
1.
0.05 M HCl: dilute concentrated HCl (11.6 M) with Milli-Q dH2O (4.3 mL of conc. HCl/L solution) and sterile filter.
-
2.
Mouse collagen IV (Collaborative Biomedical Products; cat. no. 354233): dilute to desired concentration with 0.05 M HCl; 1 μg collagen/cm2 dishes are used for passaging keratinocytes and 5 μg collagen/cm2 dishes are used for induced differentiation and/or protein extraction (see Note 5 ).
-
3.
D-PBS with added 1% antibiotic/antimycotic (see Subheading 2.2. , item 7 for description of antibiotic/antimycotic).
-
4.
Tissue culture dishes (Corning, cat. no. 430165 or 430166): 35-mm or 60-mm tissue culture-treated culture dishes (polystyrene, sterile).
2.4 Keratinocyte Isolation and Passage
2.4.1 Primary
-
1.
Use newborn (d 0–2) mice (see Note 2 ).
-
2.
100×15-mm Bacteriological Petri dish (see Subheading 2.2. , item 3).
-
3.
Whatman Grade no. 1 filter paper (see Subheading 2.1. , item 2).
-
4.
70% EtOH.
-
5.
D-PBS with added 1% antibiotic/antimycotic (see Subheading 2.2. , item 7 for description of antibiotic/antimycotic).
-
6.
EMEM.06 (see Subheading 2.2. , item 9).
-
7.
CM1 (see Subheading 2.2. ).
-
8.
Humidified incubator at 37°C, set to 4.5% CO2 (see Note 6 ).
-
9.
Collagen IV-coated culture dishes at 1 μg collagen/cm2 or 5 μg collagen/cm2 (see Subheadings 2.3. and 3.3. ).
-
10.
Polystyrene serological pipets (Falcon; BD Biosciences, Franklin Lakes, NJ; cat. nos. 357543, 357551, and 357525).
-
11.
HEPES buffered saline, pH 7.4 (HBS: ref. 3): 7.14 g HEPES Powder (30 mM), 0.72 g dextrose (d-glucose, anhydrous, 4 mM), 0.22 g KCl (3 mM), 7.60 g NaCl (130 mM), 0.142 g Na2HPO4 (1 mM); 0.0012 g Phenol Red; ∼14 mL 1 M NaOH. Add all dry chemicals to 600 mL Milli-Q dH2O, mix well; add 14 mL 1 M NaOH, mix, and adjust to pH 7.4; bring to 1 L final volume; sterile filter.
-
12.
Mouse keratinocyte growth medium (N-medium): Use 1∶1 CM1 and EMEM.06 (see Note 7 ); N-medium with the following additives is good for 4–5 d at 4°C.
-
13.
0.25% Trypsin (see Subheading 2.2. , item 11).
2.4.2 Additives
-
1.
Epidermal growth factor (EGF; BD Biosciences, cat. no. 354001): dissolve in sterile solution of 0.1% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO; cat. no. 40001) and HBS; make suggested working solution of 10 μg/mL EGF and add at 1 μL/5 mL (or 2 ng EGF per mL of N-medium); freeze at −20°C in small aliquots; stable for up to 2 yr; avoid freeze-thaw cycles.
-
2.
Aminoguanidine nitrate (AG; MW 137.1; Sigma-Aldrich; cat. no. A5,610-8): dissolve in endotoxin-free dH2O, sterile-filter; make suggested working solution of 0.75 M and add 1 μL/mL (for final concentration of 0.75 mM in N-medium); store at 4°C, heat to 37°C, and vortex 10 s to use; make fresh every 6–8 wk.
-
3.
Cholera toxin (CT; Vibrio cholerae, Type Inaba 569B; Calbiochem, San Diego, CA; cat. no. 227035): dissolve in HBS; make suggested stock solution of 10−6 M; from this make a suggested working solution of 10−7 M and add 1 μL/mL (for final concentration of 10−10 M in N-medium); store aqueous solution at 4°C for 6–8 mo.
-
4.
Hydrocortisone (HC; minimum 98%; Sigma-Aldrich; cat. no. H-4001): make suggested working solution of 0.04 mg/mL HC in HBS as follows; dissolve HC in absolute EtOH at the rate of 10 mg HC/1 mL EtOH, then add HBS to bring to volume; sterile filter; add 10 μL/mL of working solution to N-medium; store working solution at 4°C for up to 1 yr.
2.4.3 Subculture
-
1.
0.25% Trypsin (see Subheading 2.2. , item 11).
-
2.
D-PBS (see Subheading 2.1. ).
-
3.
EMEM.06 (see Subheading 2.2. , item 8).
-
4.
N-medium (see Subheading 2.4. ).
-
5.
Humidified incubator at 37°C, set to 4.5% CO2.
-
6.
Collagen IV-coated culture dishes at 1 μg collagen/cm2 or 5 μg collagen/cm2 (see Subheadings 2.3. and 3.3. ).
-
7.
Polystyrene serological pipets (see Subheading 2.4. , item 7).
2.5 Shipping and Receiving Mouse Skins
2.5.1 Shipping
-
1.
Washing medium: D-PBS with added 1% antibiotic/antimycotic; 0.1% gentamicin reagent solution (50 mg gentamicin sulfate/mL distilled water; Invitrogen/GIBCO; cat. no. 5750060).
-
2.
Transport medium: Dulbecco’s modified Eagle medium containing 1000 mg/L d-glucose, l-glutamine, Pyridoxine HCl, 110 mg/L sodium pyruvate, 3.7 g/L sodium bicarbonate, pH 6.7 (low-glucose DMEM; Invitrogen/GIBCO; cat. no. 31600-034); 20% FBS; 1% HC (of 0.04 mg/mL suggested working solution, or 0.4 μg/mL final volume; see Subheading 2.4.2. , item 4); 1% antibiotic/antimycotic; 0.1% gentamicin reagent solution.
-
3.
70% EtOH.
-
4.
15 mL and 50 mL Sterile centrifuge tubes (polyethylene terephthalate; Corning; cat. nos. 430055 and 430304).
2.5.2 Receiving
See Subheading 2.4. for keratinocyte isolation and passaging supplies.
2.6 Freezing and Thawing Viable Stocks
2.6.1 Freezing
-
1.
0.25% Trypsin (see Subheading 2.3. , item 11).
-
2.
Mouse keratinocyte freezing medium (mKFM): EMEM without calcium chloride; 8.33% v/v dimethyl sulfoxide (DMSO) (endotoxin-free sterile-filtered, MW 78.13; Sigma-Aldrich; cat. no. D2650); 20% cFBS. Light-sensitive; prepare the day of use.
-
3.
95% EtOH at room temperature.
-
4.
3–4 lbs Dry ice.
-
5.
Vermiculite-insulated beaker and polyurethane-skinned foam ice bucket (see Fig. 1 ): Insulated beaker consists of a 600-mL beaker inside a 1000-mL beaker; the space between the two is filled with vermiculite, and the top of the space is sealed with paraffin (see Note 8 ).
-
6.
Filter (embedding) bags (approximate size 2.5×2.75 in.; made of lens paper; suspended in 95% EtOH with open paper clip or similar wire).
-
7.
Liquid nitrogen freezer, −196°C.
-
8.
Polystyrene serological pipets (see Subheading 2.4.1. , item 10).
-
9.
1.8 mL NUNC freezing vials, internal thread (Nalge Nunc International; cat. no. 368632): use 1 per 106 cells.
2.6.2 Thawing
-
1.
One need the items under Subheading 2.4.1. , items 8–12 plus 70% EtOH).
2.7 Induction of Differentiation Markers
-
1.
D-PBS (see Subheading 2.1. , item 4).
-
2.
1 M CaCl2 solution (see Subheading 2.2. , item 2).
-
3.
EMEM.06 (see Subheading 2.2. , item 9).
-
4.
Keratinocyte growth medium with 0.15 mM Ca2+ (KGM.15): Keratinocyte Growth Medium (KGM; BulletKit® without Ca2+; Cambrex Bioscience, Walkersville, MD; cat. no. CC-3104; see Note 9 ); 1 M CaCl2 solution, added 0.15 μL/mL, to bring medium to 0.15 mM Ca2+. Store KGM.15 for 1 mo at 4°C/darkness.
3 Methods
Whenever possible, procedures should be conducted in a Type IIA laminar flow hood to minimize the risk of contamination.
3.1 cFBS (1)
3.1.1 Rinsing Chelex 100 Resin (see Note 1 )
-
1.
Add 1.3 L Milli-Q dH2O to flask or beaker.
-
2.
Add 200 g Chelex 100 Resin to Milli-Q dH2O while stirring.
-
3.
Adjust pH to 7.35–7.4 with HCl (3–6 M).
-
4.
Turn off stirrer and let Chelex settle for 30 min.
-
5.
Pour off dH2O while being careful not to pour off any Chelex.
-
6.
Wash two times with 1.3 L of Milli-Q dH2O: add dH2O, stir 5 min, let Chelex settle for 30 min, then pour off dH2O.
-
7.
Wash two times with 1.3 L of D-PBS as in step 6. On the last wash with D-PBS, stabilize pH to 7.35–7.4 before letting Chelex settle.
-
8.
Pour off PBS carefully.
3.1.2 Preparing Chelexed Serum
-
1.
Add 1 L of cold (4°C) FBS.
-
2.
Stir 1 h at 4°C in darkness (wrap with aluminum foil).
-
3.
Filter cFBS once through a 0.45-μm filter to remove remaining resin and again through a 0.22-μm filter to sterilize.
-
4.
Set aside a sample to determine Ca2+ concentration (see Note 4 ).
-
5.
Label, aliquot into 15-mL or 50-mL sterile containers and store at −20°C/darkness (see Note 10 ).
3.2 CM1
The following protocol assumes 30–40 mice will be used, yielding 900–1200 mL CM1. Amount of collagenase solution may need to be modified if using less than 30 mice.
3.2.1 Day 1
-
1.
Obtain live, newborn mouse pups.
-
2.
Decapitate using sterile razor blade, rinse in 70% EtOH, and remove limbs and tail (see Note 11 ).
-
3.
Slice skin ventrally from neck to tail and peel off in one piece.
-
4.
Rinse skins in three changes of D-PBS with added 2% antibiotic/antimycotic to prevent contamination. Skins can stay submerged in D-PBS solution at room temperature for up to 1 h.
-
5.
Place precut sterile Whatman Grade no.1 filter paper into 100-mm Petri dish and spread intact skin directly onto dry paper, dermal side down. Make sure edges of the skin are not rolled. Five to six skins can be spread on one 100-mm Petri dish.
-
6.
Add 7–10 mL of cold 0.25% trypsin per dish.
-
7.
Refrigerate 16–18 h at 4°C (see Note 12 ).
3.2.2 Day 2
-
1.
Separate epidermis from dermis with fine sterile forceps: Epidermis is shiny, relatively thin, and transparent whereas dermis is red, relatively thick, and opaque (see Note 13 ).
-
2.
Place dermis pieces in a 60-mm Petri dish with 5 mL of 37°C collagenase solution (see Note 14 ).
-
3.
Mince dermis with sterile scissors and transfer to sterile Erlenmeyer flask.
-
4.
Add 45 mL of 37°C collagenase solution and stir uncovered in a 37°C incubator set to 5.0% CO2 for 30 min (see Note 15 ).
-
5.
Filter through sterile gauze into sterile centrifuge tubes to remove dermis pieces.
-
6.
Centrifuge for 5 min at 200g to pellet cells. Carefully aspirate supernatant.
-
7.
Resuspend in HCM. Plate 1 skin/T150 tissue culture flask in 25 mL of HCM (see Note 16 ).
-
8.
Culture at 37°C in 5% CO2 for 1 d (see Note 17 ).
3.2.3 Day 3
-
1.
Wash flask two times with D-PBS to remove traces of calcium.
-
2.
Feed with 30 mL of EMEM.06 and culture at 37°C in 5% CO2 for 2 d.
3.2.4 Day 4
Incubate.
3.2.5 Day 5
-
1.
Filter CM1 from each flask through 0.45-μm 500-mL bottle-top filters (see Note 18 ).
-
2.
Aliquot into tubes or bottles for storage at −20°C. Keep frozen until needed, avoiding multiple freezes/thaws (see Note 19 ).
3.3 Coating Culture Dishes With Mouse Collagen Type IV
-
1.
Thaw vial of mouse collagen type IV slowly; place vial in container of ice and place container at 4°C (thawing should take approx 24 h). Vortex vigorously for 15 s to fully dissolve.
-
2.
Calculate ug collagen per cm2 using the μg/mL information provided by the manufacturer. Use 3 mL of collagen solution per 60-mm dish as the standard (see Note 20 ). In a laminar flow hood, dilute to the desired concentration with sterile 0.05 M HCl.
-
3.
Add working collagen solution to tissue culture dishes at the rate of 1 μg/cm2 to passage, or 5 μg/cm2 to induce differentiation and/or to harvest cells for protein extraction. Allow dishes to sit in the laminar flow hood for 1 h.
-
4.
Remove working collagen solution using sterile technique and save for future use (see Note 21 ).
-
5.
Rinse each dish two times using D-PBS with added 1% antibiotic/antimycotic to remove the acid. Remove as much D-PBS as possible after the final rinse by tilting the dish and aspirating thoroughly.
-
6.
Use immediately or wrap dishes in plastic wrap—several may be grouped together—and store at 4°C for up to 2 yr.
3.4 Keratinocyte Isolation and Passage
3.4.1 Primary
-
1.
Skin newborn mouse pups and float on 0.25% trypsin overnight at 4°C, as described in Subheading 3.2.1. (see Note 22 ).
-
2.
Separate epidermis from dermis with sterile forceps.
-
3.
Place epidermis in sterile centrifuge tube with EMEM.06 (EMEM.06 should be at room temperature): 1–4 skins: use a 15-mL tube with 5–10 mL of medium; 5+ skins: use a 50-mL tube with 10–20 mL of medium.
-
4.
Shake tube(s) firmly approx 50 times to separate cells (see Note 23 ).
-
5.
Remove large piece(s) of epidermis with a sterile instrument.
-
6.
Count cells in a hemacytometer and centrifuge at 200g for 5 min.
-
7.
Aspirate supernatant. Mouse keratinocytes will adhere to glass; using a plastic pipet, resuspend in appropriate volume of N-medium to plate at the following seeding densities on collagen-coated dishes:
-
1.0×106 cells/60-mm dish in 5 mL of N-medium
-
0.45×106 cells/35-mm dish in 2.5 mL of N-medium (see Note 24 ).
-
-
8.
Incubate at 37°C, 4.5% CO2 (see Note 6 ). Feed every other day with 5 mL of N-medium/60 mm dish and 2.5 mL of N-medium/35-mm dish, or every third day with 6 mL of N-medium/60-mm dish and 3 mL of N-medium/35-mm dish (e.g., for a 35-mm dish, feed 2.5 mL on Monday, passage on Wednesday, and feed 2.5 mL, then feed 3 mL on Friday). Do not exceed 2 d without feeding.
-
9.
Passage or raise calcium (see Subheading 3.7. ) when cells are completely confluent (every 7–8 d).
3.4.2 Subculture
-
1.
Wash gently with D-PBS.
-
2.
Add 0.25% trypsin as follows: 2 mL/35-mm dish; 3 mL/60-mm dish.
-
3.
Incubate at 37°C for 6–8 min.
-
4.
Using a plastic pipet, triturate with an equal volume of cold (4°C) EMEM.06 to dislodge cells and dilute trypsin. Remove to a cold centrifuge tube (see Note 25 ).
-
5.
Count cells in a hemacytometer and centrifuge 3–4 min at 200g.
-
6.
Aspirate supernatant and, using a plastic pipet, resuspend in appropriate volume of N-medium to plate at the following seeding densities on collagen-coated dishes: 0.5 m 106 cells/60-mm dish in 5 mL of N-medium; 0.2×106 cells/35-mm dish in 2.5 mL of N-medium.
-
7.
Incubate at 37°C, 4.5% CO2. Feed as in the primary protocol ( Subheading 3.4.1. , step 8) described previously.
-
8.
Passage again or raise calcium when cells are completely confluent (every 7–8 d; see Note 26 ). Cells can be subcultured indefinitely; however, expression of late epidermal differentiation markers, such as profilaggrin and keratin 1, are lost by the 10th passage (4).
3.5 Shipping and Receiving Mouse Skins
3.5.1 Shipping
-
1.
Skin newborn mouse pups as described in Subheading 3.2.1. , steps 1–4.
-
2.
Rinse skins in three changes of washing medium to prevent contamination. Skins can stay submerged in washing medium at room temperature for up to 1 h.
-
3.
Fill one 15-mL centrifuge tube for each skin with transport medium kept at 4°C. Transport medium should fill the tube to the top to ensure coverage of the entire skin. (Leave a small bubble of air to compensate for potential pressure changes en route.)
-
4.
Place each skin in a separate tube of transport medium. Cap tightly and seal with parafilm.
-
5.
Place tubes in styrofoam mailing container with ice packs and added insulation, such as styrofoam peanuts. Ship by overnight express. Notify recipient of impending arrival.
3.5.2 Receiving
-
1.
Carefully open tubes and note any shipping damage (see Note 27 ).
-
2.
Remove skins from transport medium with a sterile instrument and rinse in several changes of D-PBS with added 1% antibiotic/antimycotic to remove residual FBS (calcium).
-
3.
Process as described in Subheading 3.4.
3.6 Freezing and Thawing Viable Stocks
3.6.1 Freezing (see Fig. 1 )
-
1.
Trypsinize cells as described in Subheading 3.4.2. , steps 1–5.
-
2.
Aspirate supernatant and, using a plastic pipet, resuspend in mKFM at 106 cells/mL. DMSO in mKFM may be toxic to cells, especially at room temperature or above, so do not allow cells to linger in freezing medium (no more than 30 min before the cooling procedure is started).
-
3.
Transfer cell suspension into freezing vials at 1 mL/vial (106 cells/vial recommended freezing density). Seal cap well, but not too tightly or container may burst during subsequent thawing.
-
4.
Place no more than three vials upright in each 2.5×2.75 in embedding bag.
-
5.
Place stir bar in insulated beaker, place beaker in ice bucket; place ice bucket on stirring motor, and fill beaker one half to two thirds full with 95% EtOH and turn on stirring motor.
-
6.
Suspend embedding bags filled with vials by bent paper clips resting on the edge of the freezing beaker. It is best to use no more than four embedding bags, with three vials per bag (or 12 total vials at once). Only submerge the cell suspension, the cap of the vial should be completely above the ethanol level.
-
7.
Fill ice bucket with 95% EtOH to slightly below the ethanol level in the freezing beaker (level will rise with the addition of dry ice pellets).
-
8.
Add dry ice pellets to 95% EtOH in the ice bucket (not in the beaker; the goal is to lower the temperature inside the beaker approx 1°C/min) (see Note 28 ). Add pellets sparingly at first to avoid spill-over, but be sure that pellets are visible in the ice bucket throughout the procedure (check periodically).
-
9.
After 1 h, measure the temperature in the freezing beaker. Vials are ready to be transferred to a liquid nitrogen freezer when the temperature of the ethanol in the freezing beaker has reached −40°C or below (takes approx 1.5 h).
3.6.2 Thawing
-
1.
Put 5 mL of N-medium into a 60-mm collagen-coated dish and place in 37°C incubator set to 4.5% CO2. Allow to equilibrate for 1 h.
-
2.
Remove frozen vial from liquid nitrogen and immediately immerse in a 37°C water bath (see Note 29 ).
-
3.
As soon as the solution has thawed, wipe the area around the cap with 70% EtOH or similar antimicrobial reagent, then flick gently to resuspend cells and transfer entire contents of vial to the culture dish with a plastic pipet tip. Again, DMSO may be toxic to cells, especially at higher temperatures, so do not allow cells to linger in freezing medium.
-
4.
Feed after 24 h and resume normal feeding schedule thereafter. Cells should be ready for subculture after approx 7–10 d (when confluent).
3.7 Induction of Differentiation Markers
-
1.
Begin with confluent cells cultured in N-medium, in a dish coated with 5 μg of collagen/cm2.
-
2.
Wash gently two times with D-PBS.
-
3.
Replace N-medium with EMEM.06 at normal feeding volume to purge the cells of growth factors. Culture in EMEM.06 for 24 h.
-
4.
Refeed with KGM.15. Culture in KGM.15 for 48 h.
-
5.
Wash gently two times with D-PBS; aspirate as much buffer as possible after final rinse.
-
6.
Cells can be harvested for protein extraction or wrapped in aluminum foil and frozen at −80°C for future use (see Note 30 ).
4 Notes
-
1.
Quick chelexing method: Rather than letting the Chelex 100 Resin settle, dH2O or D-PBS can be filtered using a Buchner funnel lined with Whatman Grade no. 1 filter paper. Be sure to scrape the resin from the filter back into the flask between rinses. Do not let the resin become too dry. Quick chelexing method will result in a slightly higher Ca2+ concentration (∼0.25 mM) because of the calcium in the filter paper, but the results are still acceptable.
-
2.
Species does not seem to matter. Culture of keratinocytes from transgenic mice does not require medium conditioned by fibroblasts from genetically identical mice (i.e., normal mouse fibroblasts can be used to make CM1).
-
3.
For example, FBS with 0.134 mg/mL calcium (concentration provided by manufacturer): because FBS is added at 7.34%, final Ca2+ concentration of HCM is 0.25 mM. Make a 1 M CaCl2 working solution, sterile-filter, and add 0.35 μL/mL to HCM for a final Ca2+ concentration of 0.6 mM. CaCl2 working solution can be stored at 4°C for 2 yr if sterility is maintained and container is sealed tightly to prevent evaporation.
-
4.
When using cFBS, Ca2+ concentration can be determined by atomic absorption spectroscopy; linearity in the University of Washington Medical Center Clinical Laboratory extends to 0.009 mM. Please note that the laboratory should be educated about the nature of the specimen to avoid standard dilutions used for determination of serum concentrations. A cFBS Ca2+ concentration of approx 0.25 mM is normal.
-
5.
Testing multiple lots of collagen is recommended for best results. We test our collagen by ordering two to four distinct lots from the manufacturer, coating dishes, and carrying mouse epidermal keratinocytes in such dishes for two to three passages, taking note of cell attachment. We choose the lot that gives the best cell attachment, determined by visual inspection of Giemsa-stained colonies.
-
6.
Mouse epidermal keratinocytes are best maintained at 4.5% CO2. We recommend using an external CO2 analyzer, such as a Fyrite® Gas Analyzer (Bacharach, Inc., Pittsburgh, PA; cat. no. 10-5000), to confirm digital CO2 incubator readings.
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7.
To save time, a 2X solution of additive (2XE) can be made. 2XE is made with twice the volume of additives (20 μL/mL HC, 2 μL/mL AG, 2 μL/mL CT, 0.4 μL/mL EGF) in EMEM.06 and should be added to CM1 1∶1 to make N-medium. Store at 4°C for up to 2 wk.
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8.
Try to keep the paraffin seal dry because moisture between the layers may cause the beakers to break.
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9.
We purchase KGM without calcium from Cambrex Biosciences, then add our own calcium chloride. If small amounts of medium are to be prepared, KGM kit supplements provided by the manufacturer should be aliquoted to minimize freezing and thawing.
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10.
The shelf life of cFBS is unknown; however, our stocks have been successfully used for up to 2 yr when stored at −20°C/darkness and thawed no more than three times.
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11.
Providone iodine is toxic to keratinocytes and should not be used in this procedure. Antimicrobial preparations aside from those listed in this chapter have not been tested.
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12.
Placing the dishes on a wire shelf in the refrigerator tends to work better than a flat glass or plastic shelf. Be sure that the refrigeration unit is operating as close to 4°C as possible. The 16- to 18-h refrigeration time is crucial to successful epidermal separation.
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13.
Epidermis should come off easily, usually in one solid piece. We suggest gripping the dermis with one pair of forceps and using another to peel off the epidermis. Variation of temperature, incubation time, and enzyme used can result in an epidermis that is difficult to remove. If the skins have been in enzyme too long, basal cells are lost and gummy strands of DNA are observed.
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14.
Dermis pieces from up to 40 mice can be placed in the described amount of collagenase solution. If the epidermis is needed for keratinocyte harvest, dermis pieces can be stored in D-PBS while working with the epidermis first (see Subheading 3.4.1. , steps 3–9). When the epidermal cells are in the incubator, transfer dermal pieces to the collagenase solution and continue the CM1 protocol.
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15.
We place a stir motor inside a humidified incubator (37°C, 5.0% CO2) and thread the power cord through an opening in the top of the incubator to plug in. Be sure to use a sterile stir bar (autoclave or submerge in 70% EtOH to sterilize).
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16.
HCM is used only for plating fibroblasts and ensures that no keratinocytes are left to proliferate in the culture. Calcium concentration should be accurate (0.6 mM). You may add one extra flask for every 10–15 skins when plating fibroblasts to ensure the cells are not too confluent when EMEM.06 is added (e.g., 33 flasks for 30 mouse skins).
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17.
Fibroblasts grow very quickly. The goal is to collect conditioned medium from cells in active growth phase, so be sure to proceed with the d 3 protocol when cells are approx 60% confluent (i.e., check cells early in the morning and do not delay the procedure).
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18.
Approximately 150 mL of CM1 will go through one filter. Remember to use sterile technique throughout the procedure. Letting the empty flask stand upright for 1 min will allow 1–2 mL of residual medium to pool at the bottom and be pipetted out.
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19.
CM1 can be stored and used successfully for up to 2 yr. We have not systematically tested CM1 beyond 2 yr. Solution may separate after thawing; shake until color is homogeneous.
-
20.
Example: Corning 60 mm tissue culture dishes have a surface area of 21 cm2. Approximately 3 mL of collagen solution is sufficient to evenly cover the bottom of such a dish. You will need a working collagen solution of 105 μg/3 mL (35 μg/mL) to coat the dish at 5 μg/cm2, the suggested concentration for setting up a culture for protein extraction. Cells cultured on 5 μg collagen/cm2 cannot be passaged successfully; use a 1 μg collagen/cm2 concentration for cells to be passaged.
-
21.
Solution can be kept at 4°C on ice for up to one week before it will need to be frozen again at −80°C. Thawing more than three times is not recommended.
-
22.
For a transgenic mouse litter, it may be necessary to keep each mouse skin separate, both on trypsin and while harvesting/plating keratinocytes, until the individual mice are genotyped. The amputated tail may be taken at the time of sacrifice to verify genotype.
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23.
Holding the tube, flick sharply against palm of the hand and repeat approx 50X. Three 15-mL tubes or two 50-mL ones can be shaken at once. A low cell count may be remedied by more vigorous shaking (to adequately dislodge cells from the epidermal sheet) or by confirming enzyme (trypsin) activity. A cell count of 2–3×106 cells/skin is normal.
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24.
Large numbers of floating cells are normal, especially in the early passages.
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25.
This method does not completely inactivate trypsin, and cells should not linger in the solution. Do not use serum on the cells. We have not tested the effect of soybean trypsin inhibitor on mouse keratinocytes.
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26.
Mouse epidermal keratinocytes should be passaged only when confluent, unlike human epidermal keratinocytes, which should be subconfluent when passaged.
-
27.
If the package was not insulated well enough for the shipping conditions, medium might be partially frozen or warmed to ambient temperature, resulting in cell death. Also, international shipping may cause a day or two of transport delay, which could lead to low cell viability. You may decide to increase seeding density to compensate, or use a Trypan blue viability test when counting cells harvested from skins.
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28.
Although this freezing method is optimal (5), other conventional cryofreezing techniques—such as placing freezing vials in a tightly closed polyurethane container at −80°C overnight before transferring to liquid nitrogen storage—have yielded acceptable results. Best results occur when care is taken to lower the temperature of the cell suspension 1°C/min (down to between −40°C and −80°C) before storing vials over liquid nitrogen.
-
29.
To guard against rupture, place vial inside a latex glove or put lid on water bath. Do not completely submerge vial; be sure only the cell suspension is under water.
-
30.
The shift from 0.06 mM Ca2+ to 0.15 mM Ca2+ will induce expression of some epidermal differentiation markers. Noninduced control dishes may also be harvested using steps 5 and 6 (3).
References
Brennan, J. K., Mansky, J., Roberts, G., and Lichtman, M. A. (1975) Improved methods for reducing calcium and magnesium concentrations in tissue culture medium: application to studies of lymphoblast proliferation in vitro. In Vitro 11, 354–360.
Dulbecco, R. and Vogt, M. (1954) Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99, 167–182.
Tsao, M. C., Whithall, B. J., and Han, R. G. (1982) Clonal growth of normal human epidermal keratinocytes in a defined medium. J. Cell Physiol. 110, 219–229.
Hager, B., Bickenbach, J. R., and Fleckman, P. (1999) Long-term culture of murine epidermal keratinocytes. J. Invest. Dermatol. 112, 971–976.
Kang, S. J. and Fleckman, P. (1995) Convenient computer colony image analysis using an ordinary flatbed scanner for evaluating keratinocyte cryopreservation methods (abstract). J. Invest. Dermatol. 104, 612.
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© 2005 Humana Press Inc.
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Pirrone, A., Hager, B., Fleckman, P. (2005). Primary Mouse Keratinocyte Culture. In: Turksen, K. (eds) Epidermal Cells. Methods in Molecular Biology™, vol 289. Humana Press. https://doi.org/10.1385/1-59259-830-7:003
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DOI: https://doi.org/10.1385/1-59259-830-7:003
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