Isolation, Purification, and Cultivation of Murine and Human Keratinocytes

Abstract

The architecture of mammalian skin incorporates an outer layer of stratified epithelium. This enables the organism to conserve internal homeostasis and maintain protection from adverse environmental exposure. The keratinocyte is the cell primarily responsible for this structure. Isolation and in vitro cultivation of this cell type is widely used in dermatological and other investigations as opposed to using whole animals. However, this cell is very fastidious as compared to other skin cells (fibroblasts, etc.) and thus requires special procedures to obtain successful in vitro cultivation. This chapter describes the methodology required to isolate, purify, and cultivate keratinocytes to produce both monolayer and stratified cultures. The methodologies for producing cultures of keratinocytes obtained from rat skin and from human skin are described.

1 Introduction

The methodology of in vitro cultivation of both rat and human keratinocytes is outlined. Depending on an investigator’s goals, each source might offer an advantage. Experiments with inbred, syngeneic strains of rats might offer comparatively superior statistical data. However, using tissue obtained from humans could reduce extrapolation problems and bypass animal rights concerns. Rat keratinocytes are obtained from the skins of syngeneic newborn albino rats. Human keratinocytes are obtained from skin biopsies resulting from various surgical procedures. Both tissues are first processed to (1) minimize microbial contamination and (2) remove subcutaneous elements. The skin is then treated physically and chemically to obtain separation of the dermis from the epidermis. The separation procedure used results in the splitting of the skin at the junction where the basal layer of keratinocytes join with the underlining dermis. The resulting basal keratinocytes are then removed, suspended in a special isotonic solution, and purified via centrifugation on a density gradient. The purpose of this procedure is to remove the fibroblasts and cellular debris resulting from previous procedures. The purified keratinocytes are then resuspended and quantified. Precise amounts of keratinocytes are plated on various substrata depending on whether the purpose is to obtain a monolayer culture or a stratified, differentiated culture. Both culture types are maintained at 35°C, 5% CO₂ in a humidified environment. Growth of monolayer cultures is monitored using an inverted phase-contrast microscope or by standard histological procedures if grown on opaque substrata. The production of stratified, differentiated keratinocytes requires further steps and special substrata to obtain the desired epithelium. The cells are first incubated on a membrane submerged in growth medium until they form a confluent monolayer. They are then incubated at the air-liquid interface in order to encourage stratification to form an epidermal-like structure. This resulting culture can be examined using histological procedures for light and transmission electron microscopy.

The methodology involved in culturing mammalian cells is very complex. The demand for purity of materials that are used, the necessity of maintaining strict aseptic conditions, and the requirement of specific incubation procedures must all be met for successful cell cultivation. Any attempt to discuss all of these specific procedures in this chapter would be inadequate at best. However, a thorough knowledge of this methodology is essential for all investigators involved in mammalian cell cultivation. Fortunately, there are a number of manuals that have been published in which this methodology is discussed in detail. The one that these authors have used for many years is authored by R. Ian Freshney, the latest edition published in 2000 (1). Explicit descriptions of necessary and useful equipment, aseptic techniques required, and valuable tips designed to improve performance are contained in such manuals. For investigators not familiar with these procedures, the information contained in manuals of this type is a necessity. For seasoned investigators, such manuals remain very helpful.

2 Materials

• 1. Human full-thickness skin, surgically removed. Process immediately to obtain and cultivate keratinocytes.

• 2. Syngeneic albino rats, 2–3 d old. Process immediately to obtain and cultivate keratinocytes.

• 3. Biosafety cabinet equipped with a HEPA filter.

• 4. CO₂ Incubator with atmosphere controls.

• 5. Inverted phase-contrast microscope (IPCM).

• 6. Sterilizing equipment.

• 7. Water purification equipment (if needed).

• 8. Hemocytometer.

• 9. Reagent-grade chemicals only (alcohol, NaCl, buffers, etc.).

• 10. Bard/Parker surgical scalpel (no. 22), disposable, sterile (Baxter).

• 11. Plastics, sterile (tissue culture flasks, covered multiwells, dishes, etc.) (see Note 1 ).

• 12. Sterile centrifuge tubes (see Note 1 ).

• 13. Filter units for sterilization of chemicals and biologicals (Nalgene).

• 14. Sterile disposable pipets (see Note 1 ).

• 15. Trypsin, crude, 1:250, unsterile powder. Use working solutions immediately (BD-Difco Laboratories).

• 16. Trypsin, porcine pancreas, cell culture tested. Store at 4°C or −20°C (see Note 1 ).

• 17. Trypsin inhibitor, soybean, cell culture tested. Store at 4°C or −20°C (see Note 1 ).

• 18. Earle’s balanced salt solution (EBSS). Store at 4°C or −20°C (see Note 1 ).

• 19. Phosphate-buffered saline (PBS), Ca²⁺ and Mg²⁺-free.

• 20. Ethylenediaminetetracetic acid (EDTA) (see Note 1 ).

• 21. Trypan blue; cell culture tested (see Note 1 ).

• 22. Percoll™, sterile solution (Amersham Biosciences). Store at ambient temperature.

• 23. Percoll density marker beads (Amersham Biosciences).

• 24. Polycarbonate centrifuge tubes (Nalgene).

• 25. Minimum essential medium (MEM). Refrigerate or freeze working solutions until used (1 mo maximum) (see Note 1 ).

• 26. L-Glutamine, 200 mM. Store at −20°C. Use immediately after thawing (see Note 1 ).

• 27. Fetal bovine serum (FBS). Store at −20°C. Use immediately after thawing (see Note 1 ).

• 28. Insulin (IN), solution, from bovine pancreas, cell culture tested. Store at 4°C (see Note 1 ).

• 29. Hydrocortisone (HC)-cortisol. Cell culture tested. Store at 4°C (see Note 1 ).

• 30. Antibiotic/antimycotic solutions (penicillin, streptomycin, gentamycin, amphotericine B, neosporin, etc.). Store solutions at −20°C (see Note 1 ).

• 31. Epidermal growth factor (EGF), mouse natural, cell culture tested. Store working solutions at 4°C or −20°C (see Note 1 ).

• 32. Bovine pituitary extract (BPE). Store at −20°C. Use immediately after thawing.

• 33. Collagen, calf skin type I; cell culture tested; powder or solution. Store solutions at 4°C or −20°C (see Note 1 ).

• 34. Laminin (LMN). Engelbreth-Holm-Swarm rat sarcoma (basement membrane); cell culture tested. Store at −70°C (see Note 1 ).

• 35. Porous, inert membrane (13 mm in diameter) (Pall-Gelman, Millipore).

• 36. Glass fiber filter (44 mm in diameter) (Pall-Gelman).

• 37. Sable hair brush sterilized with 70% ethanol.

3 Methods

The methods described in this section include (1) the preparation of solutions and biologicals necessary for successful isolation and cultivation of mammalian cells, (2) the initial procedures for processing full-thickness epithelium received from rat and human skin to obtain viable keratinocytes, (3) procedures necessary to produce splitting of full-thickness skin into epidermis and dermis, (4) steps to remove mostly basal keratinocytes from the epidermis and dermis, (5) procedures for purification and enumeration of suspended keratinocytes, (6) cultivation of keratinocytes to produce monolayer cultures, (7) subcultivation of confluent monolayer cultures, and (8) cultivation of cells to produce multilayered differentiated cultures. The progress of the methodology used in obtaining viable keratinocytes for cultivation can be monitored using microscopic and/or histological examinations.

Strict aseptic conditions must be maintained in all procedures for preparing keratinocytes for eventual in vitro cultivation.

3.1 Preparation of Chemical and Biological Solutions Necessary for the Processing and Subsequent In Vitro Cultivation of Basal Keratinocytes

A number of important solutions must be carefully prepared in order to successfully process the skin for isolation, purification, and subsequent cultivation of basal keratinocytes. Because of the delicate nature of living mammalian cells, they must be suspended in solutions with the proper osmolality in order to maintain cell membrane integrity. Fortunately, most of these solutions can be purchased fully prepared for immediate use. Some can be obtained at higher concentrations (10X, 100X) for better storage and handling. If sterilization is necessary, it can be accomplished using filters of various sizes and configurations depending on the solution, its volume, and its characteristics (1). The websites of the companies listed in Subheading 2. (see Note 1 ) usually contain descriptions of the products that they sell.

3.1.1 Isotonic Solutions Used in Washing Cells and in Dissolving Solid Substances

These materials can be purchased as sterile solutions in various concentrations or as powders to be dissolved in purified water and sterilized. The exact formulations can be found in cell culture manuals or in descriptions supplied by the company.

• 1. EBSS. This solution is specially formulated to maintain cell membrane integrity. It has various uses in producing and maintaining cell suspensions for short periods prior to actual cultivation.

• 2. PBS (1–10X strength). This isotonic solution is used mostly as a solvent for dissolving various substances to be used in cell preparation procedures prior to cultivation. It is not used to store cells for extended periods. It may be necessary to include glucose (0.01% [v/v]) in this solution (PBSG).

3.1.2 Special-Purpose Solutions Necessary for Suspending, Counting, and Dissociating Keratinocytes

• 1. Trypan blue. This is the dye most frequently used in the dye exclusion test when it is necessary to determine the viability of isolated mammalian cells. The powder is usually dissolved in PBS to obtain a 0.1% solution. Small samples of cell suspensions are then added and the test performed without asepsis.

• 2. EDTA. This chemical is used to dissociate cell clumps into single cells and to dislodge cultivated cells from the substratum to result in a suspension. A concentration of 0.02 % in PBS is usually employed for those purposes. It can also be included in solutions containing other chemicals when appropriate.

• 3. Trypsin. This is the enzyme of choice for isolating keratinocytes from full-thickness skin. Solutions in EBSS or PBS ranging from 0.1% to 0.25% are used for epidermal-dermal separation. Also, it can be dissolved in EBSS or PBS to obtain solutions of 0.03% to be included with 0.02% EDTA in detaching monolayer cultures for subcultivation.

• 4. Trypsin inhibitor. It might be necessary to inhibit further trypsin enzymatic activity after a selected incubation period. A solution of soybean trypsin inhibitor at a concentration of 1 mg/mL dissolved in PBS or EBSS is used for this purpose. Complete growth medium, described in Subheading 3.1.3. can also be used to inhibit trypsin activity.

3.1.3 Basal Growth Medium Specifically Formulated for the Growth of Basal Keratinocytes

Because of the fastidious characteristics of basal keratinocytes, careful attention must be made in selecting the proper medium and specific supplements in order to promote both attachment and growth of these cells in vitro. The growth medium is the same for both rat and human cultures. It contains supplements shown to be required for optimal cell viability and proliferation of basal keratinocytes. The basal medium is MEM, which contains an exacting balance of amino acids, vitamins, inorganic salts, and glucose (1). Supplements are added to it to obtain optimal cultivation.

• 1. Hormonal supplements have been shown to affect growth control of cells in culture (2). The combination of HC and IN was shown to support proliferation of keratinocytes (3). Thus, basal medium is supplemented with both HC and IN at 10 µg/mL.

• 2. Both EGF (4) and BPE (5) have been identified as stimulating keratinocyte proliferation in culture. EGF at 10 ng/mL and BPE at approx 60 µg/mL is added to the basal medium.

• 3. Antibiotics and antimycotics are necessary supplements that control microbial contamination during long-term cultivation. Penicillin (100 units/mL) and streptomycin (100 µg/mL) are used as antibiotics in MEM. These reagents are unstable and, thus, freshly prepared solutions of these reagents should be added to growth medium weekly. Either fungizone (0.25 µg/mL) or amphotericin B (25 µg/mL) are used as antimycotics. These components form the basal MEM that support keratinocyte cultivation. It does not include animal serum supplements.

• 4. The unstable amino acid L-glutamine (200 mM) at a concentration of 0.29 mg/mL is added to the basal medium and is necessary for medium stabilization. Fresh medium containing this reagent should be used for no more than 1 wk after preparation. Thawed L-glutamine can be added to older medium at the prescribed concentration at weekly intervals.

• 5. To obtain complete growth medium (CMEM), FBS is added to the MEM with supplements to obtain a 10% (v/v) solution. It is necessary to pretest each lot of serum purchased from suppliers to confirm its effectiveness in supporting keratinocyte cultivation. Although there have been reports of successful growth of keratinocytes in vitro using serum-free medium, we find that basal keratinocytes, especially of human origin, require serum-supplemented medium for optimal attachment and proliferation. However, media can be purchased from suppliers that affirm optimal growth of keratinocytes without any animal components (Cascade Biologies; see Note 1 ). The preparation of 500 mL of MEM is outlined in Table 1 .

Table 1 Ingredients Needed for 500 mL of Complete Minimum Essential Medium

3.1.4 Growth Factors That Enhance the Attachment and Thus Subsequent Growth of Keratinocytes on Various Substrata

The surface of commercial tissue culture vessels are prepared to promote attachment and growth of mammalian cells in vitro. However, keratinocytes are more exacting in their requirement for optimal proliferation. Two sub-stances included in the basement membrane of stratified epithelium have been shown to enhance attachment of keratinocytes (i.e., collagen [6] and laminin [7]). Various substrata used in culturing basal keratinocytes, such as plastic and glass culture vessels or synthetic membranes, should be precoated with these substances.

• 1. We have experienced best results with LMN at 1 mg/cm² of surface. Aliquots of the purchased stock solution of LMN, containing 1 mg/mL in buffered NaCl, are added to the culture surface and are allowed to evaporate to dryness in a biosafety cabinet. Coated culture vessels should be used within 5–7 d after evaporation of NaCl.

• 2. Calf skin collagen type 1 is applied at 6–10 µg/cm² of surface (8). A purchased solution of collagen (1 mg/mL) is diluted to 50 µg/mL with 0.1 N acetic acid. The required amount is plated onto the surface and incubated for 1 h at room temperature. The surface is then rinsed with PBS to remove the acid and air-dried in a biosafety cabinet. Coated culture vessels should be used within 5–7 d after drying is complete.

3.2 Preparation of Full-Thickness Skin for Basal Keratinocyte Isolation

This subsection details steps necessary to (1) minimize microbial contamination of the tissues obtained from rat and human biopsies and (2) remove as much subcutaneous and dermal tissue as possible so as to facilitate epidermal-dermal separation. Because to the considerable differences in the anatomical structure of newborn rat skin as compared to human skin, procedures for the preparation, and treatment of the two skin types to obtain basal keratinocytes must be described separately. Also, keratinocytes obtained from individual human skins, both neonatal and adult, cannot be successfully pooled for subsequent cultivation as can skin from syngeneic rat littermates.

3.2.1 Preparation of Full-Thickness Skin From Inbred Laboratory Rats for Basal Keratinocyte Isolation (see Note 2 )

The inbred CFN albino rat was found to be a convenient and accessible source of rat keratinocytes. The skins from animals 24–36 h old were found to give the best results in subsequent epidermal-dermal separation. One litter usually consists of 10–15 animals.

• 1. The animals are killed by cervical dislocation and the total body cleansed using cotton soaked with 70% ethanol.

• 2. To minimize variations in skin thickness, only the backs of the animals are used. With surgical scissors, the back skin from the nape of the neck to the beginning of the tail is dissected. The resulting skin tissues measure approx (2−3) × (4−5) cm, depending on the age of the animals.

• 3. Any loose subcutaneous elements are carefully removed with a scalpel and discarded. The processed skins can be placed in a dish containing EBSS or PBS before proceeding to the next step.

3.2.2 Human Skin Obtained From Surgical Procedures

Human skin, handled aseptically, can be obtained from various sources, including neonatal foreskin and skins resulting from cosmetic surgery. Most of the adult tissues received are the result of breast reduction and the removal of abdominal skin after weight loss.

• 1. The skins are handled aseptically in a biological safety cabinet with filtered positive air pressure. The objective is to eliminate possible microbial contamination resulting from preparation procedures.

• 2. All subcutaneous elements, containing mostly adipose tissue, are removed and discarded.

• 3. The full-thickness skin is placed, dermal side down, in a 150-mm plastic dish and covered with PBS (minus Ca²⁺ and Mg²⁺) containing the antibiotics described in Subheading 3.1.3. , step 3. They remain in the dish for 30–40 min before the solution is removed.

• 4. An attempt is then made to remove as much of the underside of the dermis as possible to facilitate dermal-epidermal separation. To accomplish this, the skins are placed, epidermal side down, in a 150-mm dish cover and the surface of the dermis scraped with a scalpel. The purpose is to reduce the thickness of the dermis to approx 3–5 mm.

• 5. The processed tissue is then cut into sections approx 4–5 mm wide and 3–4 cm long using a scalpel.

3.3 Separation of the Dermal and Epidermal Layers of the Skin for Subsequent Harvesting of Basal Cell Keratinocytes

This procedure is designed to promote separation of the two anatomical layers where basal keratinocytes in the epidermis are attached to the surface of the dermis. The objective is to obtain as many viable basal cells as possible. Experimental studies indicate that only these cells are capable of proliferation in vitro (9). Both physical and chemical steps are incorporated in producing epidermal-dermal separation. It has been reported that stretching skin biopsies promotes this separation (10) and that trypsinization at lower temperatures results in less cell damage (11). This subsection describes epidermal-separation procedures for both rat and human keratinocytes. These steps and all subsequent steps are performed under strict aseptic conditions in biological safety cabinets.

3.3.1 Treatment of Rat Skin Samples to Obtain Dermal-Epidermal Separation

• 1. The prepared skin samples are washed with fresh EBSS chilled to 4°C. Three individual samples are then placed, stratum corneum down, on the surface of 100-mm plastic tissue culture dishes.

• 2. Each sample is held down with forceps and, using a scalpel, scraped laterally from the center with force in order to cause the skin to stretch and adhere to the bottom of the dish.

• 3. The adhering samples are then chilled to 4°C in the refrigerator and a sterile stock solution of cold 1:250 trypsin at 0.25% (w/v) in EBSS or PBS carefully added to completely cover the skins. Incubation in the trypsin is continued at 4°C for 14–16 h depending on the age of the litter (see Note 3 ).

3.3.2 Treatment of Human Skin Samples to Obtain Dermal-Epidermal Separation

• 1. The narrow skin strips described in Subheading 3.2.2. are placed dermal side down in a large plastic tissue culture dish (100–150 mm) prechilled to 4°C to promote adhesion of the skin to the bottom of the dish.

• 2. Powdered trypsin is dissolved at room temperature in EBSS to obtain a 0.1–0.13% solution. It is then sterilized using a 0.2-µm filter unit (1) and chilled to 4°C in the refrigerator.

• 3. Approximately 50 mL of the chilled trypsin solution is added to the adhering skins in a 150-mm dish (less for smaller dishes). Incubation of the tissue in the enzyme is continued at 4°C for 12–18 h or until the epidermis and dermis can be physically separated (see Note 4 ).

3.4 Obtaining Keratinocytes From the Separated Epidermis and Dermis

This subsection describes the methodology required to harvest basal keratinocytes from the skin after splitting its two main layers. Steps must be taken to protect the viability of the cells that have been subjected to very harsh physical and chemical procedures. As the process of isolating and suspending cells progresses, resulting samples of cell suspensions can be monitored microscopically using IPCM.

3.4.1 Suspension of Rat Basal Keratinocytes Obtained From the Separated Epidermis and Dermis into Stabilizing Medium

• 1. After the enzymatic treatment, the trypsin solution is aspirated from the 100-mm dish and the tissues washed two to three times with approx 10 mL of EBSS to remove residual trypsin.

• 2. The enzymatic activity of the trypsin is neutralized by adding 10–20 mL of CMEM to each dish and allowing it to remain for 2–5 min before removal.

• 3. Using two sterile forceps, individual samples are removed and placed, stratum corneum down, in a plastic tissue culture dish. The dermis is then removed by grasping its edges with the forceps, lifting it from the epidermis, and placing it, epidermal side up, next to the exposed epidermis.

• 4. Fresh CMEM is then added to the dishes to cover the surfaces of both skin layers, and keratinocytes are carefully liberated into the medium by applying delicate strokes with a sterile fine sable hair brush across the tops of the skin layers. The sable brush was sterilized by immersion in 70% ethanol for 15–30 min, followed by washing with EBSS or PBS to remove the ethanol.

• 5. The resulting cells suspended in CMEM from all dishes are combined in one container to be processed further.

3.4.2 Suspension of Human Basal Keratinocytes Obtained From the Separated Dermis into Stabilizing Medium

The procedure for collecting human cells is identical to the one described in Subheading 3.4.1. for rat cells.

3.5 Purification and Enumeration of Basal Cell Keratinocytes Collected From the Surface of the Dermis and Epidermis

The methodology described in this subsection applies to both rat and human basal keratinocytes. Purification and enumeration must be accomplished before cell cultivation can proceed.

3.5.1 Purification of Basal Keratinocytes to Remove Fibroblasts and Cellular Debris

The cells harvested from the separated skin elements include various cell types and cellular debris resulting from the physical and chemical procedures described.

• 1. The cells collected from the tissue samples are first consolidated in conical centrifuge tubes and centrifuged at 30g for 5 min at 4°C for initial purification. This removes most of the tissue and cellular debris.

• 2. The cell pellet is gently resuspended in 5 mL of EBSS.

• 3. Added to a Nalgene centrifuge tube are 6.6 mL EBSS, 0.8 mL of 10X PBS, and 7.6 mL Percoll. The 5-mL cell suspension is then added to the tube and the contents mixed thoroughly by inverting the tube two or more times.

• 4. A continuous gradient is formed by centrifuging the resulting 38% Percoll at 30,000g for 15 min at 4°C (12,13). See Fig. 1 for the location of density marker beads and the various cell layers in the density gradient. The identities of the cells in the bands can be determined by viewing inocula on slides and cover slips via phase-contrast microscopy (see Fig. 2 and Note 5 ).

• 5. With the aid of a sterile Pasteur pipet connected to a vacuum, all of the Percoll and cellular components above the lower band of the gradient (containing the basal cells) are aspirated and discarded.

• 6. The basal cells in the lower band are collected using a 5.0-mL pipet attached to a controlled pipetting device, suspended in CMEM (8–9 mL) and centrifuged in a 10-mL graduated centrifuge at 16g for 10 min at 4°C.

• 7. CMEM is then added to the pellet to result in exactly 10 mL of packed cells and medium.

• 8. The cells are carefully and uniformly suspended into the CMEM by slow, repeated filling and emptying a 10-mL pipet.

• 9. The resuspended suspension should be enumerated immediately.

Fig. 1.

Percoll density gradient compartmentalization of cellular components resulting after keratinocyte isolation from skin samples. (A) Nalgene tube showing the location of three marker beads resulting after gradient formation; (B) Nalgene tube showing the location of cellular components resulting after gradient formation. Differentiated cells (spinous, granular, etc.), fibroblasts, and debris are located between densities of 1.075 and 1.087 g/cm³. Basal keratinocytes are located proximally at the 1.087-g/cm³ density.

Fig. 2.

Phase-contrast micrographs of cellular components separated in a Percoll density gradient and resuspended in a supporting medium. (A) Cells resuspended from the 1.075- to 1.087-g/cm³ density. Most of the cells are identified as differentiated cells and fibroblasts plus some basal cells. (B) Cells collected from the 1.087-g/cm³ density are almost exclusively basal keratinocytes (×240). Cultivation of cell suspensions from the two bands in CMEM can be used to confirm that the rounded cells in the upper band are predominantly fibroblasts, whereas the lower band contains basal cells almost exclusively.

3.5.2 Enumeration of Purified Basal Keratinocytes

The percent of viable cells in the total cell number in a suspension can be determined visually using an IPCM and a hemocytometer (1). Only viable cells are able to attach and proliferate in vitro. Therefore, the percent of viable cells in the total cell count must be determined in the attempt to produce consistent seeding inocula. The trypan blue exclusion test is employed for this purpose. The blue dye will not stain cells that are actively metabolizing and have intact membranes.

• 1. In a small tube, add 200 µL of the cell suspension to 600 µL of PBS and then add 200 µL of the 0.1% trypan blue solution and mix gently but thoroughly by inverting the tube three to five times.

• 2. With a cover slip in place on each side of the hemocytometer, add a small amount of the cell suspension to fill each chamber using a Pasteur pipet, filling via capillary action (do not overfill).

• 3. Count the entire chamber on both sides and determine the average cell number.

• 4. The following formula can be used for determining the total number basal cells in a suspension (1): average cell number per grid (mm²) × 10⁴ × dilution factor (5) = number of cells per milliliter in the suspension. Approximately (1−1.5) × 10⁸ cells can be obtained from the skins of a litter (10–15) of rats. Approximately (1.0−1.2) × 10⁸ cells can be obtained from one human skin sample 25 cm² in size, whereas approx (2.2−6.5) × 10⁵ cells can be obtained from one foreskin.

• 5. The percent viability of the cell suspension is expressed as the number of cells unstained by Trypan blue per 100 cells counted.

3.6 Establishing Monolayer Cultures of Basal Keratinocytes

Successful in vitro cultivation of isolated, purified basal keratinocytes depends on specific procedures and environmental conditions. Such considerations include (1) substratum for initial attachment, (2) initial plating density, (3) temperature, and (4) atmosphere.

3.6.1 Establishing Monolayer Cultures of Rat Basal Cells

• 1. The purified and enumerated cells are centrifuged at 16g for 10 min at 4°C and resuspended in CMEM to obtain a cell suspension containing approx 5 × 10⁵ cells/mL. We have observed that a 0.2% suspension (v/v) of rat basal cells (pellet) will result in a suspension of similar composition.

• 2. Various culture vessels are seeded with this inoculum in amounts that satisfy the working volume and surface area (see Note 6 ).

3.6.2 Establishing Monolayer Cultures of Human Basal Cells

The purified and quantified basal keratinocytes described in Subheading 3.5. are used to produce monolayer and multilayer basal keratinocyte cultures.

• 1. Experimental results in our laboratory have shown that plating approx 2 × 10⁵ purified, viable cells per square centimeter of cultivation surface results in optimal attachment and proliferation of human keratinocytes. Other investigators may prefer different initial seeding densities to obtain desired results.

• 2. The cell suspension in CMEM is pipetted into culture vessels in amounts that satisfy the working volume and surface area. For examples of steps in seeding various culture vessels with the desired amount of cells (see Note 6 ).

3.6.3 Incubation of Rat and Human Basal Cells to Establish Monolayers

• 1. The seeded cells are placed in an incubator maintained at 35°C with the atmosphere set at 95% air-5% CO₂ with a humidity of 95%. Rat and human cells respond similarly using this procedure.

• 2. Incubation is allowed to proceed for 18–24 h to allow attachment of the cells to the surface.

• 3. The original medium, containing unattached cells and possible debris, is aspirated and discarded.

• 4. An equal volume of fresh CMEM, warmed to 35°C in a water bath, is then added and incubation continued.

• 5. Every 2 d, the old medium is removed from the culture and fresh medium (warmed to 35°C) added. Cell attachment and growth is monitored daily using an IPCM. Typical cultures at various stages are shown in Fig. 3 .

Fig. 3.

Phase-contrast photomicrographs of rat basal keratinocytes seeded onto collagen-coated plastic substrata. Soon after plating (18–24 h), the cells have attached to the substratum as shown in (A). Also shown in this figure are the results of cell proliferation in 2 d (B) and 3 d (C). Cells firmly connect to each other and form a continuous sheet constituting a confluent monolayer in 5–7 d (D) (original magnification: ×235).

3.7 Subcultivation of Primary Monolayers of Keratinocytes

Primary cultures of human basal keratinocytes can be subcultured for use in experimentation. This is best accomplished when the primary culture reaches 70 to 80% confluence. Primary cells obtained from one culture vessel can be subcultured into two vessels of similar surface area.

• 1. The growth medium (CMEM) is first removed from the selected culture, which is then washed with a solution containing 0.02% EDTA in PBS minus Ca²⁺ and Mg²⁺.

• 2. Next, a solution containing 0.03% trypsin and 0.01 % EDTA in PBS is added to the culture vessel to cover the cells.

• 3. Incubation proceeds at 37°C for approx 2 min or until the cells detach from the surface of the culture vessel as determined via microscopic examination.

• 4. Further enzymatic activity is inhibited by adding an equal or greater volume of CMEM or an equal volume of soybean trypsin inhibitor dissolved in PBS (1 mg/mL).

• 5. The detached cell suspension is transferred to a centrifuge tube and centrifuged at 16g for 5 min at room temperature.

• 6. The cells are resuspended in fresh CMEM resulting in an amount that will produce the 1:2 split. Cultivation of the seeded, passed cells follows the described procedures for keratinocyte growth in vitro.

3.8 Methodology for Constructing a Differentiated, Stratified Keratinocyte Culture In Vitro

A stratified differentiated keratinocyte culture developed in vitro using isolated basal cells might be advantageous in some experimental designs (8,14,15). Such a culture is produced by incubating cultured keratinocytes at the air-liquid interface (16).

• 1. The initial seeding, as described in Subheading 3.6. , is on a porous, inert membrane that is autoclave sterilized. We have used the Puropore membrane supplied by Gelman (Ann Arbor, MI) with a pore size of 0.2 µm and a diameter of 13 mm (see Note 7 ).

• 2. To enhance attachment, the membrane should be pre-coated with laminin or collagen using the procedure for coating culture vessels (see Subheading 3.1.4. ).

• 3. The coated membranes are then placed in 24-well plastic tissue culture vessels and seeded with 1 × 10⁶ cells/cm². At least two coated wells are seeded in the absence of a membrane so that the attachment and proliferation of the culture can be monitored using IPCM.

• 4. The cell inoculum seeded on the coated membranes is incubated submerged in CMEM for 4–5 d as described for monolayer cell cultivation (see Note 8 ). Verification of the attachment and growth of the cells on the membrane can be accomplished via histological staining procedures and light microscopy (1). A stained 5-d-old culture of human keratinocytes on a membrane is shown in Fig. 4 .

  The next step is to raise the porous membrane to the air-liquid interface.

• 5. Glass fiber filters 44 mm in diameter, sterilized via autoclave, are placed in 60-mm culture dishes and CMEM is added to accomplish complete saturation of the filters without excess fluid.

• 6. Up to five Nylon membranes (two is optimal) previously coated, seeded, and incubated for the 4- to 5-d period are transferred to the surface of the saturated filters. They are positioned so that the surface containing the cultured keratinocytes is exposed to the atmosphere rather than covered with medium and fed with CMEM via contact with the saturated filter below (see ref. 9).

• 7. Incubation of the lifted cultures is continued for an additional 10–15 d while being fed fresh medium three times per week. This is done by aspirating as much of the old medium as possible and adding fresh CMEM to resaturate the filter.

• 8. The resulting stratification and differentiation can be observed using histological procedures for preparing sections for light microscopy and for transmission electron microscopy (TEM). Such observations have verified the development of a stratified epithelium complete with stratum corneum. Ultrastructural markers characteristic of normal mammalian skin also develop in the lifted culture. Using TEM methodology, these structures have been observed in both rat (7) and human (13) lifted cultures.

Fig. 4.

Light photomicrograph of a 5-d culture of human basal keratinocytes on a collagen-coated porous membrane. The membrane containing the cells was stained with hemotoxylin and eosin using histological procedures (original magnification: ×150).

4 Notes

• 1. These companies are the prime suppliers of mammalian cell culture products. Most of the products listed can be purchased from more than one source. Also, many of the reagents listed can be purchased in various forms, including dry and lyophilized powders, frozen in ampules, and as ready-to-use or concentrated solutions. They can also be purchased in various combinations to fit specific cultivation procedures. Descriptions of these products can be obtained at the company’s websites or in brochures: Sigma-Aldrich (http://www.sigmaaldrich.com/); Invitrogen (http://www.invitrogen.com/); BD Biosciences (http://www.bdbiosciences.com/); BD (http://www.bd.com/ds/); Cellgro (http://www.cellgro.com/); Falcon Labware (http://www.bacto.com.au/falcon.htm); Corning (http://www.corning.com/); ICN Biomedicals (http://www.icnbiomed.com/); Cascade Biologies (http://www.cascadebiologics.com).

• 2. The newborn CFN albino rats from our breeding pens average 10–15 animals per litter. Smaller litters (i.e., those with less than 10 animals) tend to produce larger, more developed animals and are not used. Newborn rats can also be obtained from laboratory animal suppliers such as Charles River Laboratories (Wilmington, MA). Litters of animals purchased from suppliers can be shipped along with the nursing doe or shipped under other arrangements.

• 3. We began cultivating rat keratinocytes in 1971. At that time, the trypsin available was the crude powder (1:250) supplied by Difco Laboratories (Detroit, MI). A number of purified trypsin enzymes are now available and much less toxic. Investigators should explore these substances, which will most likely give better results. Because of our ongoing experimentation using rat keratinocytes, we did not introduce such a change in our procedure, which might make comparisons with earlier experimental data invalid.

• 4. We use the same procedure for splitting human skin and rat skin. However, because of the advanced development of human skin, results similar to those of newborn rat skin cannot be duplicated. First, it is very difficult to obtain a comparable degree of stretching with the firm human skin and, second, enzymatic treatment under cold conditions does not produce the ease of epidermal-dermal separation observed with newborn rats. As a result, skin splitting of most human skin samples following cold enzymatic treatment is often difficult to obtain. The strength of the trypsin solution and the incubation time selected for enzymatic treatment depend on the thickness of the individual skin specimens. There are, undoubtedly, other variations in individual human skin samples that could impact on the enzymatic separation. Skin characteristics differ depending on the age, gender, and physical condition of the donor. Also, the postoperation time of skin storage before being processed must be considered. These variations might necessitate adjustments in the trypsinization procedure for dermal-epidermal separation. An increase in the strength and incubation time of enzymatic treatment might obtain better separation results. However, this also produces an adverse affect on cell viability. The degree of separation should be checked prior to removing the enzyme from the dish. If, with the use of forceps, the epidermis can be lifted from the dermis but is still connected at the center of the skin strips, then, in our experience, this results in an optimal separation. However, if the separation is already complete and the epidermis has floated from the dermis, there has been overtrypsinization. In the former case, the resulting quantity of isolated basal cells might be less but the quality as observed in cell viability, cellular attachment, and proliferation will be more satisfactory. Experience obtained from processing different human skins is the only solution to this problem.

• 5. It might not be possible to remove all fibroblasts from suspensions of basal keratinocytes using the Percoll gradient. However, this purification step is necessary in order to obtain a successful cultivation of the latter cell type. Comparatively small numbers of fibroblasts remaining in suspensions can eventually outgrow the keratinocytes in areas, dislodging them from the culture. Therefore, the more fibroblasts that can be removed, the more uniform the resulting mono-layer of keratinocytes will be.

• 6. The proper seeding of the purified and quantified basal cell keratinocytes to obtain the desired number of cells per square centimeter of substratum surface can be accomplished in a number of ways. One example in seeding cells at 2 × 10⁵ cells/cm² of culture surface could include the following steps in the procedure: First, if a total of 1 × 10⁸ cells were found to be obtained from a 25-cm² human skin sample (or from the skins of 12 newborn rats) following purification and enumeration, they could be resuspended in 10 mL of CMEM to result in a stock suspension containing 1 × 10⁷ cells/mL. The exact amount of cells could then be seeded into vessels of various surface sizes. Because a T75 flask has 75 cm² of culture surface and a working volume of 15 mL, a total of (2 × 10⁵)(75) or 150 × 10⁵ cells are needed (i.e., 1.5 mL of the stock suspension). Pipetting 1.5 mL of the stock suspension into the flask plus 14.5 mL of additional CMEM completes the procedure. Other culture vessels could be seeded with this stock suspension depending on their culture surface and working volume. Another example would be more appropriate if only one vessel type is to be seeded. T75 flasks can be seeded with 15 mL of a stock suspension containing 2 × 10⁶ cells. It must be pointed out that the culture surface and working volume of vessels obtained from various suppliers could differ.

• 7. We have used Gelman’s nylon membrane (Puropor-200), which is no longer available, almost exclusively. Other membranes of similar pore size might produce similar results. Glass fiber filters were used because of their ability to become saturated with medium. Other such filters of similar inert structure could be just as effective. Selections can be made from companies manufacturing porous membranes (Gelman, Millipore, etc.).

• 8. The methodology developed for producing stratified cultures of human keratinocytes results in variations not observed with rat keratinocytes. This is most likely the result of the differences in human samples as described in Note 4 . We have been able to produce somewhat better results by modifying the CMEM supplements. The FBS is increased to 15% and BPE doubled to approx 120 µg/mL.