Isolation and Culture of Skin Fibroblasts

Abstract

Dermal fibroblasts synthesize and organize skin connective tissue (dermis). Fibroblast cultures provide a powerful tool for investigating both the molecular mechanisms that regulate skin connective tissue production and the pathophysiology of fibrotic skin diseases. In this chapter, we describe detailed procedures for establishing and maintaining primary cultures of adult human fibroblasts. We also provide protocols for constructing three-dimensional dermal and skin equivalent culture models.

1 Introduction

Cultivation of fibroblasts in vitro provides a useful model for studying their functions in normal and pathological states in a controlled environment. Several decades ago, cell culture techniques were considered somewhat esoteric. Today, because of better understanding of cell nutrition, metabolism, and general growth requirements, cell culture has become a routine procedure.

A good cell culture model is one in which the cells faithfully and reproducibly mimic their behavior in their natural in vivo environment. The extent to which these criteria are met is often difficult to access and therefore, caution should always be exercised when extrapolating observations made on cultured fibroblasts to in vivo settings. Nevertheless, fibroblast cultures have proven to be useful tools for understanding molecular mechanisms that regulate fibroblast function. To obtain meaningful data, it is important that culture systems are standardized and maintained in appropriate conditions in order to minimize culture artifacts. In this chapter, we provide detailed protocols for cultivation of primary adult human skin fibroblast used in our laboratory, starting with isolating cells from adult skin. Protocols are given for cultivation of fibroblasts in monolayer on tissue culture dishes, in three-dimensional collagen lattices, and in co-culture with human keratinocytes to form so-called skin reconstructs.

1.1 Fibroblasts in Human Skin

Skin is one of the most suitable tissues that can be used as a source of human fibroblasts. Skin consists of three layers: (1) epidermis, which primarily contains keratinocytes, melanocytes, and Langherans cells; (2) dermis, primarily populated with fibroblasts, blood vessels, and dendritic cells; and (3) subcutaneous tissue. Epidermis is separated from dermis by a basement membrane, which is primarily composed of extracellular matrix proteins (collagens and laminins), synthesized by both fibroblasts and keratinocytes.

Type I collagen is the most abundant protein in the dermis. It is produced by resident fibroblasts, which also synthesize other components of dermal extracellular matrix, including other collagens (III, V, VII), elastin, proteoglycans, and fibronectin. In human skin, the half-life of type I collagen is estimated to be greater than 1 yr (1). Collagen synthesis is increased after a wound, to remodel the injured area (2,3), and in fibrotic diseases such as scleroderma (4,5). Collagen synthesis is decreased after sun exposure and during aging (6). Fibroblasts also produce extracellular matrix-degrading enzymes such as matrix metalloproteinases (MMPs) and plasmin. Homeostasis of extracellular matrix depends on balanced synthesis of all these proteins.

Skin is particularly adaptable to organ cultures. Skin organ cultures have been used for decades to study skin ex vivo (7). Organ cultures allow the use of reagents, which are not approved for human use or not compatible with topical application, to study skin function. However, organ culture is not suitable for the study of individual cell types, as is cell culture.

1.2 Collagen and Fibroblast Culture

In monolayer cultures, numerous reports have shown that proliferation and collagen production of human skin fibroblasts depend on the composition and pH of the medium, the source and concentration of serum, the addition of ascorbate, and the age of the donor. It has also been shown that collagen synthesis per cell decreases with increased cell confluence (8).

1.3 Primary Human Skin Fibroblast Cultures

Normal human dermal fibroblast cultures can be divided into three phases:

• 1. Primary cultures are established by enzymatic digestion of the dermis, or by outgrowth of fibroblasts from explanted tissue pieces. It is important to note that primary cultures are not derived from a single cell, but rather are a heterogeneous mixture of skin fibroblasts.

• 2. Secondary cultures are actively proliferating cells, obtained by passage and expansion of primary cultures. After a limited number of cell divisions (30–50 cycles for fibroblasts from skin of young adults), the rate of cell growth gradually decreases.

• 3. Terminal cultures eventually reach a state of replicative senescence, considered to be aging at the cellular level (9,10).

One advantage of studying fibroblasts obtained from primary culture is that they contain normal diploid complement of chromosomes, and therefore, in this respect, mimic fibroblasts in vivo. In contrast, fibroblast cell lines typically have numerous chromosomal aberrations and mutations, which are associated with their ability for unlimited growth.

1.4 Culture of Fibroblast in Monolayers

When grown in monolayer culture in their proliferative phase, fibroblast cultures expand, and require subculturing to alleviate overcrowding. If the cell density is not reduced at confluence, cells will detach from their support and die.

Cryogenic preservation of low passage-number fibroblasts is useful for maintaining reserves of cells. In doing so, it is easy for one investigator to rapidly build a bank of fibroblasts from a limited number of skin samples.

1.5 Culture of Fibroblasts With Collagen

In skin, fibroblasts are in contact with type I collagen and other extracellular matrix components. These contacts are critical for normal regulation of fibroblast function. Therefore, culture of fibroblasts on a collagen-coated surface or embedded in collagen gel is commonly done to model in vivo environment.

Culture of skin fibroblasts in collagen gels (also called collagen lattices or dermis equivalents), was first described by Bell et al. (11). The introduction of fibroblasts into a three-dimensional collagen matrix leads to a reorganization of the matrix by fibroblasts. This reorganization is readily observable as reduction in the size of the gel resulting from fibroblast contraction of the collagen fibrils. Thus the collagen gel serves as a stimulus to modulate fibroblast behavior (12). When cultured in collagen lattices, fibroblast morphology and many aspects of their behavior differ from those observed in monolayer cultures. Morphologically, fibroblasts in monolayer are flat, spindle-shaped, and organized in parallel arrays. When cultured in a three-dimensional collagen gel, fibroblasts are elongated and have numerous dendrites. Fibroblasts in collagen gels proliferate much slower than in monolayer cultures. Metabolic activities also differ, for example synthesis of collagen is reduced (13,14) and MMP activities are increased (15,16) in collagen gels.

Variations of collagen gel models have been developed to assess fibroblast behavior within a three-dimensional matrix. In noncontracting (or attached) collagen lattices, contraction of collagen occurs in the vertical axis but is limited in the other dimensions. Fibroblasts in noncontracting collagen gels spread and elongate but are unable to reorganize the collagen fibers. Downregulation of collagen is less pronounced than in contracted collagen gels. The degree of gel contraction reflects the level of mechanical tension on the cells. Mechanical tension influences a wide array of cellular functions (17,18).

1.6 Skin Equivalents

Skin equivalents consist of fibroblasts embedded in a collagen gel, or dermal equivalent, covered with an epidermis equivalent composed of several layers of differentiated keratinocytes. In contrast with monolayer cultures and collagen gels, skin equivalents include keratinocyte-fibroblast interactions in a physiologically and spatially organized mode. The culture is maintained at the air-liquid interface, allowing formation of a stratified epidermis, which exhibits partial barrier function (19). Skin equivalent models enable the exposure of fibroblasts to topically applied drugs or lights through the epidermis layers, and thereby allow investigation of keratinocyte modulation of fibroblast responses in a collagen gel.

Skin equivalents were originally obtained by growing differentiated keratinocytes on devitalized pigskin dermis (20). Since then, many substrates have been used to support the growth of fibroblasts and keratinocytes, including fibroblast-populated collagen lattices (21,22). Besides type I collagen, artificial dermis may contain additional extracellular matrix proteins including type III or IV collagens and proteoglycans. The degree of complexity of skin equivalents can be further increased by adding other cell types to the culture (e.g., melanocytes, inflammatory cells, cancer cells).

2 Materials

• 1. Dermal skin fibroblasts are cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% serum (fetal bovine or calf) and antibiotics and antimycotics (optional—see Notes 1–4). Supplemented culture medium will be called complete medium.

• 2. Fibroblasts are adherent cells that must be cultured in tissue culture type dishes. For culture maintenance purposes, 75-cm² dishes are commonly used (see Note 5).

• 3. Volumes stated in the following protocols are for 75-cm² dishes; they can be proportionally reduced or increased for culture vessels of other sizes.

2.1 Obtaining Fibroblasts

• 1. Punch biopsies are obtained from volunteer’s non-sun exposed buttock skin. Punch biopsies are 4 to 6 mm in diameter (see Notes 6 and 7).

• 2. Skin samples are collected in Hank’s balanced salt solution (HBSS), supplemented with penicillin (100 IU/mL), streptomycin (100 µg/mL), and amphotericine B (0.25 µg/mL) (see Note 8).

2.2 Skin Organ Cultures

Skin organ cultures are maintained in DMEM-HamF12 1:1 containing 3.75 mM glutamine, 1.8 mM CaCl₂, and 2% serum.

2.3 Primary Culture of Fibroblasts

2.3.1 Enzymatic Digestion of Dermis

• 1. 0.25% Trypsin (w/v) in phosphate-buffered saline (PBS).

• 2. Sterile forceps.

• 3. Ethanol sterilized nylon mesh.

• 4. Enzyme solution: N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid [HEPES] containing Richter’s improved MEM insulin medium [RPMI], supplemented with 1 mM sodium pyruvate, 2.75 mg/mL bacterial collagenase, 1.25 mg/mL hyaluronidase, and 0.1 mg/mL DNase I.

2.3.2 Outgrowth of Fibroblasts From Explants

• 1. 150-mm sterile dish (type Pyrex®).

• 2. Storage medium: HBSS supplemented with antibiotics and antimycotics.

• 3. Sterile scalpel and forceps.

• 4. Explantation medium: DMEM containing 20% serum, antibiotic/antimycotic supplement.

2.4 Subculture of Fibroblasts

• 1. Trypsin-ethylenediamine tetraacetic acid (EDTA) solution: 0.05% (w/v) trypsin in PBS containing 1 mM EDTA (see Note 9).

2.5 Freezing Fibroblasts

• 1. Freezing medium: DMEM containing 20% serum and 10% dimethylsulfoxide (DMSO).

• 2. Cryogenic vials.

• 3. Slow cooling rate device: programmable electronic freezer unit (−1 to −3°C per minute) or mechanical freezer unit (Mr. Frosty™ from Nalge Nunc International, Rochester, NY; StrataCooler™ from Stratagene, La Jolla, CA).

• 4. Liquid nitrogen freezer and protective gloves and glasses.

2.6 Culture of Fibroblasts With Collagen

Important: see Note 10 for handling collagen solutions.

2.6.1 Collagen Coating

• 1. Type I collagen solution (BD Biosciences, San Jose, CA) diluted to 250 µg/mL with 18 mM acetic acid.

• 2. 24-Well plates.

• 3. 2% (w/v) serum albumin solution in PBS, sterilized by filtration.

• 4. Sterile PBS.

2.6.2 Collagen Lattice Cultures

• 1. “Bacterial type” Petri dishes. The method is described for 35-mm diameter dishes. Stated volumes can be proportionally increased for culture dishes of bigger sizes.

• 2. Stock solution of 2.5X DMEM: DMEM prepared from powder, sterilized by filtration, and stored at 4°C up to 1 mo. This stock solution is used to counteract dilution of medium obtained by addition of collagen (solubilized in acetic acid) and sodium hydroxide (used to neutralize acetic acid).

• 3. Lattice medium: for 1.25 mL per 35-mm diameter dish, mix 0.70 mL 2.5X DMEM, 0.26 mL distilled H₂O, 0.09 mL 0.1 M NaOH, and 0.20 mL serum.

• 4. Type I collagen solution (BD Biosciences) diluted to 2 mg/mL with 18 mM acetic acid.

• 5. 18-Gage needles and 1-mL syringes for collagen and cell suspension pipetting make the manipulation easier (they can easily be placed back in the tube between two samples).

2.6.3 Collagenase Digestion for Isolation of Cells

• 1. Bacterial collagenase solution: dilute bacterial collagenase from Clostridium histolyticum (Sigma, St. Louis, MO) to 1 mg/mL in collagenase buffer (50 mM Tris, 0.15 M NaCl, 4 mM CaCl₂, pH = 7.4) before each use. Prepare 1 mL per lattice in microtubes (see Note 11).

2.7 Skin Equivalents

• 1. 24-mm diameter culture inserts with a 0.4-µm pore sized polyester membrane, and placed in individual wells of 6-well plates (Costar, Corning Inc, Acton, MA). The clear membrane allows observation of the culture with an inverted microscope.

• 2. Type I collagen solution (BD Biosciences) diluted to 3 mg/mL in 0.18 mM acetic acid.

• 3. 2.5X DMEM, 0.1 M NaOH (see collagen lattice protocol).

• 4. Human adult keratinocytes needed for establishing skin equivalents can be obtained from primary cultures of skin samples (23). Human adult keratinocytes are also commercially available either as cryopreserved vials or proliferating cultures (Cascade Biologics, Portland, OR; BD Bioscience-Clontech, Palo Alto, CA). Keratinocytes are handled according to the protocol provided with the cells.

• 5. Compositions of the different culture media used for skin equivalents are detailed in Table 1 . Stock solutions for making media can be stored up to 3 mo at indicated temperatures. Once prepared, skin equivalent media must be sterilized by filtration and can be stored up to 2 wk at 4°C.

Table 1 Composition of Skin Equivalent (SE) Media

3 Methods

3.1 Obtaining Fibroblasts

• 1. Thoroughly wash the biopsy site with an antiseptic soap and swab with 70% ethanol. Ethanol should be allowed to completely air-dry before the biopsy is taken. A local anesthetic (e.g., 1% lidocaine) is administered prior to biopsy. The anesthetic should be injected adjacent to the biopsy site rather than directly into it.

• 2. Stabilize the skin with the thumb and forefinger, stretching it slightly perpendicular to the normal skin tension lines.

• 3. Place the punch instrument perpendicular to the skin and apply firm and constant downward pressure with a circular motion, avoiding back-and-forth twisting motions. A definite “give” occurs when the punch instrument reaches the subcutaneous fat, indicating that a full thickness cut has been made.

• 4. Remove the punch instrument and apply downward pressure at the sides of the wound. Gently elevate the core, using aseptic forceps, and excise it at its base using small scissors. While applying pressure on the wound in preparation for closure, store the biopsy into a sterile vial containing storage medium.

• 5. Wounds 3 mm or less can be treated with a hemostatic agent and allowed to heal freely, whereas larger wounds require one or two sutures (in this case, no hemostatic agent is needed).

After removal, skin is able to withstand prolonged storage in the cold, simplifying the time schedule for culture preparations. Successful cultures can be made from material stored up to 48 h at 4°C.

3.2 Skin Organ Cultures

Skin is particularly adaptable to organ culture methods. However, small biopsies (2 mm) should be used to allow sufficient penetration of nutriments and oxygen through the entire specimen.

Skin biopsies are placed into organ culture medium immediately after removal. The culture should be started as soon as possible after biopsies are obtained.

• 1. Transfer each skin sample into a culture plate (e.g., 6-well plate) using sterile forceps, orientating the tissue so that the dermis is in contact with the bottom of the dish.

• 2. Incubate the plate 15 min at room temperature to allow good adhesion of the dermis onto the culture dish.

• 3. Gently add a small amount of medium on the side of the biopsy, making sure that the epidermis is exposed to the air. Culture up to 48 h in the 5% CO₂ incubator at 37°C.

3.3 Primary Culture of Fibroblasts

3.3.1 Enzymatic Digestion vs Outgrowth Procedures

Fibroblast primary cultures can be obtained by two methods: enzymatic digestion of dermis, or outgrowth of cells from explanted tissue pieces. The first method is faster, and yields higher recovery of cells from the skin sample. Enzymatic digestion should be utilized when fibroblast motility may be impaired (e.g., skin sample from elderly people). Although the cultures initially contain a variety of cell types, culture conditions select for fibroblast growth.

The outgrowth method relies on the capacity of fibroblasts to migrate out of the skin and adhere to the surface of the culture vessel. This method has the advantage that the migrating cells are highly enriched in fibroblasts. However, this method may also select for fibroblasts with higher rates of motility.

3.3.2 Enzymatic Digestion of Dermis

• 1. Transfer the biopsy and medium into a sterile plastic dish.

• 2. Add 10 mL of 0.25% trypsin. Incubate 30 min at room temperature.

• 3. After incubation, isolate the dermis from epidermis using sterile forceps. Cut the dermis into small pieces. Add 10 mL of enzyme solution and incubate at room temperature for 3 h.

• 4. After incubation, mechanically dissociate the tissue by pipetting up and down 10 times.

• 5. Filter the cell suspension through a sterile nylon mesh to remove tissue fragments.

• 6. Centrifuge at 400g for 10 min at room temperature.

• 7. Discard the supernatant, resuspend the cell pellet in 10 mL of complete medium and transfer cell suspension into a 75-cm² culture dish. Place cultures in the 5% CO₂ incubator at 37°C.

• 8. Change the medium 24 h later to remove nonadherent material.

3.3.3 Outgrowth of Fibroblasts From Explants

• 1. Transfer the skin sample into a 150-mm sterile dish containing 25 mL of storage medium.

• 2. Dissect the dermis from the rest of the skin (epidermis, subcutaneous tissue, vascular structures) using scalpel and forceps. The dermis can be distinguished from other structures in that it sticks to dissection instruments.

• 3. Mince the dermis into small pieces (about 1 mm³) and place about eight fragments on the bottom of a 75-cm² culture dish, separated from one another.

• 4. Allow explants to air-dry for 15 min to increase attachment to the bottom of the dish.

• 5. Gently add 8 mL of explant medium, to cover each tissue piece. Place cultures back in the 5% CO₂ incubator at 37°C.

• 6. Change the medium 24 h later and discard nonadherent material. Change the medium once per week, until substantial number of fibroblasts is observed.

Fibroblast migration out of tissue fragments should be regularly monitored using an inverted microscope. Fibroblasts should be subcultured when they occupy most of the dish surface between explants (approx 2–3 wk after start of the culture—see subculturing protocol below). After detaching cells from the dish, the explantation process can be repeated up to three times feeding the explant with 10 mL of explantation medium.

3.4 Subculture of Fibroblasts

• 1. Remove and discard culture medium.

• 2. Rinse cell layer with 2 mL of trypsin-EDTA solution to remove residual cellular debris and serum-containing medium, which contains trypsin inhibitors.

• 3. Add 3 mL of trypsin-EDTA to the cell layer and incubate at 37°C.

• 4. Observe regularly under an inverted microscope until cells are detached and float in the solution. This should occur after 2 to 5 min (if cells are difficult to detach, the incubation can be prolonged up to 10 min).

• 5. Add 5 mL of complete medium (to inactivate trypsin), gently aspirate cell suspension, and transfer it to a centrifugation tube.

• 6. Centrifuge at 400g for 10 min at room temperature (see Note 12).

• 7. Discard the supernatant, resuspend the pellet in 1 mL of complete medium, and transfer the cell suspension into a new dish containing 10 mL of complete medium.

One confluent 75-cm² dish contains approx 3 × 10⁶ cells. New dishes can be seeded at 0.75–1 × 10⁶ cells/dish.

3.5 Freezing and Thawing Fibroblasts

3.5.1 Freezing Fibroblasts

• 1. Prior to freezing, the culture should be in actively growing phase, and free from any fungal, bacterial or mycoplasma contamination.

• 2. Use previously described protocol for subculturing fibroblasts to detach and isolate the cells.

• 3. After centrifugation, remove and discard the supernatant and slowly resuspend the cell pellet with freezing medium using approx 1 mL per 75-cm² dish.

• 4. Label cryogenic vials (cell source, passage number, and date) with low temperature resistant ink.

• 5. Add 1 mL of cell suspension per vial and seal.

• 6. Place in a slow cooling rate device overnight at −80°C.

• 7. Transfer the vials to a liquid nitrogen freezer. Make sure that the location and labeling recording system is suitable for long-term cryogenic storage (see Note 13).

3.5.2 Thawing Fibroblasts

• 1. Using protective gloves and clothing, remove vials from the liquid nitrogen freezer. Update the freezer log appropriately.

• 2. Loosen the cap on each vial one-quarter turn to release liquid nitrogen from the threads, and re-tighten on the caps.

• 3. Thaw the vials by gentle agitation in a 37°C water bath, making sure to keep the cap out of the water to avoid contamination.

• 4. As soon as the content is thawed, remove vial from the water and disinfect the vial with 70% ethanol.

• 5. Transfer the vial content to a 75-cm² culture dish containing 10 mL complete medium.

• 6. Place the cultures in the incubator. Change the medium 24 h later to remove nonadherent cells.

3.6 Culture of Fibroblasts With Collagen

3.6.1 Collagen Coating

Stated volumes are for coating a 24-well plate. They can be proportionally increased or decreased for culture dishes of different sizes.

• 1. Spread 500 µL of type I collagen solution per well over the surface of a sterile 24-well plate.

• 2. Air-dry overnight in a tissue culture hood to allow complete evaporation of solution and fixation of collagen.

• 3. Incubate 1 h with 500 µL of a 2% serum albumin solution to saturate free binding sites.

• 4. Rinse three times with 500 µL of PBS before seeding fibroblasts (see Note 14).

3.6.2 Collagen Lattice Cultures

3.6.2.1 Contracting Collagen Gels

• 1. Dispense 1.25 mL of lattice medium into a 35-mm diameter dish. Incubate at 37°C until needed (step 3).

• 2. Prepare fibroblast suspension from monolayer cultures, according to subculturing protocol (Subheading 3.4.). Resuspend the resulting cell pellet in 1X DMEM. Adjust the cell density to 0.4 × 10⁶ cells/mL.

• 3. For each lattice:

  • a. Add 0.5 mL of collagen solution to the lattice medium.

  • b. Agitate the dish in a circular motion, to allow an even neutralization of collagen by NaOH (homogeneous color).

  • c. Gently mix the cell suspension and add 0.25 mL to the dish. Repeat the agitation procedure.

  • d. After cell suspension is well mixed, allow collagen to gel without disturbing, i.e., at room temperature for 15 min.

• 4. The gel should be detached from the side of the dish. If not, gently agitate the dish to detach the lattice. Place dish in the 5% CO₂ incubator at 37°C.

Culture medium should be replaced at least every week with fresh complete medium (see Note 15).

3.6.2.2 Noncontracting Collagen Gels

Prior to making noncontracting collagen gels, place an ethanol-sterilized nylon mesh, of the same diameter as the Petri dish, onto the bottom of the dish. Then, follow the same protocol as for contracting gels.

3.6.3 Collagenase Digestion of Collagen Gels for Recovery of Cultured Fibroblasts

• 1. Pre-warm bacterial collagenase solution at 37°C (1 mL/lattice in microtubes).

• 2. Transfer each lattice into the bacterial collagenase solution using forceps, and incubate in a water bath at 37°C for 10 min. Agitate the tubes after 5 min by inverting them.

• 3. When lattices are completely digested, stop the digestion by addition of 1 mM EDTA and centrifuge for 10 min at 400g at room temperature to pellet the cells.

3.7 Skin Equivalents

3.7.1 General Design

Table 2 summarizes the different steps for generating skin equivalents.

Table 2 Different Steps for Building Skin Equivalents

3.7.2 Skin Equivalent

The following protocol is described for six wells. Volumes can be proportionally changed for different quantities.

• A. D 1: Collagen gel without cells.

  • 1. Using sterile and prechilled reagents, mix on ice and in the order indicated 3.2 mL of 2.5X DMEM, 0.8 mL of serum, 0.85 mL of distilled water, 0.48 mL of 0.1 M NaOH, and 2.67 mL of 3 mg/mL type I collagen.

  • 2. Immediately mix, by gently pipetting up and down, until color is homogeneous, and immediately dispense 1.3 mL of solution per insert.

  • 3. Incubate 15 min at room temperature without disturbing to allow collagen to gel, and place the plate in the 5% CO₂ incubator at 37°C.

• B. D 2: Dermis equivalent.

  • 1. Prepare a cell suspension of fibroblasts at 245,000 cells/ml in 1X DMEM, according to subculturing fibroblast protocol (Subheading 3.4.).

  • 2. Using prechilled reagents, mix on ice and in order 7.8 mL of 2.5X DMEM, 1.95 mL of serum, 1.17 mL of 0.1 M NaOH, and 6.5 mL of 3 mg/mL type I collagen.

  • 3. Gently mix, by pipetting up and down, until color is homogeneous, and add 2 mL of the cell suspension prepared in step 1.

  • 4. Mix by pipetting up and down, and dispense 3 mL of cell suspension into each insert, on top of acellular gels.

  • 5. Incubate 15 min at room temperature without disturbing to allow collagen to gel, and place the plate in the 5% CO₂ incubator at 37°C for 7 d.

• C. D 9: Seeding keratinocytes. After 7 d, the dermal equivalent is partially contracted, to form a concave upper surface.

  • 1. Remove and discard culture medium without disturbing collagen gels, and rinse three times (5 min between rinses) with SE1 medium: 2 mL in the insert and 3 mL in the well.

  • 2. After the third rinse, remove and discard medium. Place in sterile hood during preparation of keratinocytes (approx 20 min).

  • 3. Prepare a suspension of keratinocytes at 3 × 10⁶ cells/mL in SE1 medium.

  • 4. Dispense drop-wise 100 µL of keratinocyte suspension on top of each dermal equivalent. During this step, maintain pipet as vertical as possible and tip as close as possible to the dermis equivalent. Do not try to spread the cell suspension, but rather target each drop to the center of the gel.

  • 5. Carefully place the plate back in the incubator, keeping it as horizontal as possible. Incubate for 2 h without medium to promote keratinocyte attachment to the collagen gel.

  • 6. Gently add 2 mL of SE1 medium to the insert on top of the gel, and 3 mL of SE1 medium to the well.

  • 7. Place the plate back in the 5% CO₂ incubator at 37°C.

• D. D 13: Replace SE1 by SE2 medium.

  • 1. Remove and discard medium as completely as possible without disturbing the reconstructs.

  • 2. Gently add 2 mL of SE2 medium in each insert and 3 mL of SE2 medium in each well.

  • 3. Place the plate back in the 5% CO₂ incubator at 37°C.

• E. D 15: Exposing keratinocytes to air.

  • 1. Remove and discard medium from the insert and the well.

  • 2. Add 1 mL of SE3 medium in the well; no medium is added to the insert. Placing medium only in the well allows keratinocytes to be in contact with the air, which promotes stratification and differentiation.

  • 3. Exchange medium in the well three times a week until d 21, when reconstructs are ready for experiments.

Once ready for experiments, skin equivalents can be maintained in SE3 medium for up to 1 wk. After this time, keratinocytes proliferation diminishes.

4 Notes

• 1. All techniques described must be performed in sterile conditions and proper disposal and decontamination practices should be followed. Moreover, all human samples should be considered as possible sources of infectious agents and should be handled appropriately.

• 2. Culture media are supplemented with antibiotics and antimycotics. However, antibiotics do not eliminate problems of contamination resulting from poor sterilization techniques or antibiotic-resistant micro-organisms.

• 3. In order to avoid exposure of cultures to temperature fluctuations, reagents used for cell culture should be prewarmed to 37°C, in water bath, and the container outsides should be rinsed with 70% ethanol before use, in order to prevent contamination.

• 4. It is good practice to routinely and carefully examine cultures to determine their status and health.

• 5. Adherent cells like fibroblasts must be cultured in specially treated vessels (dishes or flasks) commonly called “tissue culture” type. The bottom surface of these vessels is specially treated to enhance cell attachment and cell survival. Several vessel sizes are commercially available.

• 6. Before any material can be collected from a human volunteer, ethical approval for the research must be obtained from the local Institutional Review Board. Only trained and authorized personnel should perform skin biopsies, and every subject from whom skin is taken must give written informed consent. Furthermore, it is essential that the designation of the cell strain is unambiguous. It should be unique and maintain donor anonymity.

• 7. Using cells derived from laboratory staff for research purposes is not recommended. The use of tissue from laboratory staff for the development of transformed cell lines is prohibited, as the person concerned would have no immunity to the transformed cells.

• 8. Adult skin is invariably contaminated with micro-organisms, mainly nonpathogenic bacteria or molds. In order to insure good sterility of the tissue for cell culture, these micro-organisms must be removed as much as possible prior to skin biopsy.

• 9. Cells can also be detached using nonproteolytic methods. These methods should be used especially when use of cells with intact plasma membrane is required (adhesion studies, membrane protein extraction). To detach cells without proteolysis, use Versene buffer (0.8 mM EDTA, 0.15 M NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 1.6 mM KH₂PO₄, pH 7.2.; 20 min at 37°C) or EDTA (1–5 mM at 4°C, 20–30 min).

• 10. Type I collagen is soluble only at low temperature and low pH. It will start to gel and become insoluble if either the temperature or the pH is raised. For this reason, the stock collagen solution should be kept at 4°C until it is needed. During handling, it should be maintained at 4°C. Keep in mind that collagen will start to gel as soon as it is mixed with sodium hydroxide. Therefore, as soon as the collagen is added to the solution, the following steps should be performed as quickly as possible.

• 11. Unlike human collagenase, bacterial collagenase is able to digest mature collagen into small fragments. The main source of bacterial collagenase is Clostridium histolyticum. Collagenase preparations are rarely pure and contain other proteases, including trypsin- and chymotrypsin-like activities. Thus, to avoid cellular damage resulting from proteolysis, incubations with bacterial collagenase should be kept to the minimum time necessary to release cells from the collagen gel.

• 12. Centrifugation following trypsin detachment of cells removes trypsin and cell debris. This removal helps to maintain viability and longevity of cultures.

• 13. It is recommended to record the details concerning cell cultures, including the type, sources, and batch numbers of all media and additives, and the methods by which the cultures were established. It is helpful to record the subculture ratios and the passage number.

• 14. Collagen coatings tend to detach in long-term cultures. Alternatively, a doublelayered collagen coating can provide a stable substrate. The second layer is dispensed after the first one is completely dry.

• 15. Collagen reorganization is observed as a reduction in size of the collagen gel. Under the conditions described, contraction of the gel can be observed 2 to 6 h after seeding cells, is half complete after 24 h, and complete after 4 d. To quantify gel contraction, the diameter of the gel can be measured using a ruler on top of the dish placed on a dark background.