Establishment of Fibroblast Cultures UNIT 2 - · PDF fileEstablishment of Fibroblast Cultures...

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UNIT 2.1 Establishment of Fibroblast Cultures This unit describes methods for establishing fibroblast cultures from skin. Because fibroblasts can be expanded to relatively large numbers from a small skin sample, they have been widely used to study basic aspects of cell biology as well as genomic, biochemical, and/or functional abnormalities in human patients and in transgenic or knockout animals. Two protocols are described for the development of fibroblast lines from human and mouse skin samples; the same protocols are also applicable, with slight modification, to other animals including rats and rabbits. The first technique (see Basic Protocol) employs an “skin explant” culture system in which fibroblasts grow out of skin specimens. The second technique (see Alternate Protocol) employs a dissociated fi- broblast culture system in which fibroblasts are first released from skin specimens by enzymatic digestion and then placed in culture. In general, the explant culture system is technically simpler, requiring almost no special experience or reagents, whereas the dissociated fibroblast culture is more suitable for obtaining relatively large numbers of fibroblasts in a short period. NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4. NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. Surgical equipment may be sterilized by simply soaking in 70% ethanol; however, it is important to rinse in PBS before use since ethanol will “fix” the tissue. BASIC PROTOCOL SKIN EXPLANT CULTURE When skin specimens are “transplanted” onto culture plates, fibroblasts (in dermis) and keratinocytes (in epidermis) migrate over the plastic surfaces, as they do in an ordinary skin graft. Because fibroblasts will eventually overgrow keratinocytes in conventional culture media, relatively pure fibroblast cultures can be obtained by simply placing small pieces of skin on tissue culture dishes. On the other hand, the optional enzymatic separation of the epidermis will ensure the absence of epidermal components from the resulting fibroblast cultures; it will also allow investigators to establish keratinocyte cultures from the same skin samples. Materials Skin specimen (see Commentary) Phosphate-buffered saline (PBS; see recipe) 0.5% (w/v) dispase II (Boehringer-Mannheim) in PBS (store up to 3 months at 20°C; optional) 0.3% (w/v) trypsin (from bovine pancreas; Sigma) in PBS (store up to 3 months at 20°C; optional) Complete growth medium (DMEM or RPMI; see recipe) Trypsin/EDTA solution (see recipe) 0.4% (w/v) trypan blue in PBS (store up to 6 months at room temperature) Freezing medium: 10% DMSO/90% FBS or 10% DMSO/90% complete DMEM 15-ml and 50-ml polypropylene centrifuge tubes Lids from 100-mm tissue culture dishes Eye forceps (2 pairs) Surgical scalpel with disposable no. 22 blade Contributed by Akira Takashima Current Protocols in Cell Biology (1998) 2.1.1-2.1.12 Copyright © 1998 by John Wiley & Sons, Inc. 2.1.1 Preparation and Isolation of Cells
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  • UNIT 2.1Establishment of Fibroblast Cultures

    This unit describes methods for establishing fibroblast cultures from skin. Becausefibroblasts can be expanded to relatively large numbers from a small skin sample, theyhave been widely used to study basic aspects of cell biology as well as genomic,biochemical, and/or functional abnormalities in human patients and in transgenic orknockout animals. Two protocols are described for the development of fibroblast linesfrom human and mouse skin samples; the same protocols are also applicable, with slightmodification, to other animals including rats and rabbits. The first technique (see BasicProtocol) employs an skin explant culture system in which fibroblasts grow out of skinspecimens. The second technique (see Alternate Protocol) employs a dissociated fi-broblast culture system in which fibroblasts are first released from skin specimens byenzymatic digestion and then placed in culture. In general, the explant culture system istechnically simpler, requiring almost no special experience or reagents, whereas thedissociated fibroblast culture is more suitable for obtaining relatively large numbers offibroblasts in a short period.

    NOTE: All incubations are performed in a humidified 37C, 5% CO2 incubator unlessotherwise specified. Some media (e.g., DMEM) may require altered levels of CO2 tomaintain pH 7.4.

    NOTE: All solutions and equipment coming into contact with cells must be sterile, andproper sterile technique should be used accordingly. Surgical equipment may be sterilizedby simply soaking in 70% ethanol; however, it is important to rinse in PBS before usesince ethanol will fix the tissue.

    BASICPROTOCOL

    SKIN EXPLANT CULTURE

    When skin specimens are transplanted onto culture plates, fibroblasts (in dermis) andkeratinocytes (in epidermis) migrate over the plastic surfaces, as they do in an ordinaryskin graft. Because fibroblasts will eventually overgrow keratinocytes in conventionalculture media, relatively pure fibroblast cultures can be obtained by simply placing smallpieces of skin on tissue culture dishes. On the other hand, the optional enzymaticseparation of the epidermis will ensure the absence of epidermal components from theresulting fibroblast cultures; it will also allow investigators to establish keratinocytecultures from the same skin samples.

    Materials

    Skin specimen (see Commentary)Phosphate-buffered saline (PBS; see recipe)0.5% (w/v) dispase II (Boehringer-Mannheim) in PBS (store up to 3 months at

    20C; optional)0.3% (w/v) trypsin (from bovine pancreas; Sigma) in PBS (store up to 3 months at

    20C; optional)Complete growth medium (DMEM or RPMI; see recipe)Trypsin/EDTA solution (see recipe)0.4% (w/v) trypan blue in PBS (store up to 6 months at room temperature)Freezing medium: 10% DMSO/90% FBS or 10% DMSO/90% complete DMEM

    15-ml and 50-ml polypropylene centrifuge tubesLids from 100-mm tissue culture dishesEye forceps (2 pairs)Surgical scalpel with disposable no. 22 blade

    Contributed by Akira TakashimaCurrent Protocols in Cell Biology (1998) 2.1.1-2.1.12Copyright 1998 by John Wiley & Sons, Inc.

    2.1.1

    Preparation andIsolation of Cells

  • 35-mm tissue culture dishes or 6-well plates22-mm glass coverslips (wrap several coverslips in aluminum foil and

    sterilize by autoclaving)Hemacytometer25-cm2 tissue culture flasks1.5-ml cryotubes (e.g., Nunc)Liquid nitrogen freezer

    CAUTION: When working with human blood, cells, or infectious agents, appropriatebiosafety practices must be followed.

    NOTE: Use Milli-Q water or equivalent in all protocol steps and for preparing allsolutions.

    Prepare skin sample1. Wash skin samples in PBS by gently shaking or agitating in a 50-ml polypropylene

    centrifuge tube.

    When handling skin samples from mice, rats, or rabbits, it is easier to remove hair beforeexcision. This can be achieved by applying 70% ethanol on skin and shaving with asingle-edged razor blade (e.g., Personna Prep).

    2a. For relatively large skin samples (e.g., foreskin, surgically removed samples, orcadaver skin): Place sample on the lid of a 100-mm tissue culture dish and spread itout with the epidermal side down. Remove the subcutaneous tissue by scraping thedermal side using two pairs of eye forceps. Cut skin into strips of 0.5-cm widthusing a surgical scalpel.

    2b. For smaller skin samples (e.g., punch-biopsy samples): Place the sample on a 100-mmtissue culture dish and excise the subcutaneous tissue using a surgical scalpel and apair of forceps.

    Remove epidermis (optional)3a. For human skin samples: Incubate 45 min to 4 hr with 0.5% dispase/PBS in a 37C

    water bath, then separate as an intact sheet mechanically by gentle agitation for 10sec or by using two pairs of forceps.

    The incubation time required for epidermal separation varies depending upon the thicknessof the skin specimen and the completeness of the subcutaneous tissue removal.

    3b. For mouse, rat, and rabbit skin samples (optional): Incubate with 0.3% trypsin/PBSfor 30 to 60 min in a 37C water bath or overnight at 4C. Place the sample on thelid of a 100-mm tissue culture dish with the epidermal side up and scrape off theepidermis mechanically using two pairs of forceps.

    In general, trypsin works better than dispase for skin with deep hair follicles.

    4. Wash the dermal sample in PBS by gently shaking or agitating in a 50-mlpolypropylene centrifuge tube.

    Culture fibroblasts5. Place the dermal sample on the lid of a 100-mm tissue culture dish and cut it into

    small (2- to 3-mm) squares.

    Fibroblast outgrowth occurs only from sharply cut edges. Thus, it is crucial to use a new,fine surgical scalpel; disposable surgical blades (no. 22) can be used for this purpose.

    6. Place 5 to 10 skin pieces in the center of a 35-mm tissue culture dish or 6-well plate.

    The 6-well plates are more convenient for handling multiple fibroblast cultures in parallel.

    Current Protocols in Cell Biology

    2.1.2

    Establishment ofFibroblast

    Cultures

  • 7. Place a sterile 22-mm glass coverslip gently over the skin specimens (Fig. 2.1.1).

    Skin specimens need to be attached physically to the plate. This can be achieved mosteffectively by making a sandwich using a coverslip (Fig. 2.1.1). The coverslip also assiststhe growth of fibroblasts by maintaining the microenvironment.

    8. Add a few drops of 4C complete growth medium (DMEM or RPMI) into the spacebelow the coverslip (by applying at the edge of the coverslip so that it is drawn under),then add 1 to 2 ml of 4C complete medium to the dish or well, gently so as to avoiddisturbing the skin specimens.

    Either complete DMEM or complete RPMI can be used for growing fibroblasts.

    9. Place the culture in a humidified 37C, 5% CO2 incubator. Check the fibroblastoutgrowth every 3 to 4 days under an inverted phase-contrast microscope and changemedium every 3 to 4 days, taking care not to agitate the coverslip.

    Initial outgrowth should be detectable within 3 to 4 days (Fig. 2.1.2B). The coverslip shouldbe left on the skin specimens until the culture becomes confluent. The original skin specimensmay come off the plate spontaneously during culture and will be removed in step 10.

    10. Upon confluency, remove the coverslips and wash the dish or wells twice with 4CPBS.

    Fibroblasts sometimes migrate over the surface of the coverslip instead of the tissue cultureplate. If this is the case (as judged by focusing up and down with the microscope) transferthe coverslip into a new dish, wash it with PBS as in this step, then harvest cells by trypsintreatment as in step 11.

    Washing with PBS is essential because trypsin (see step 11) does not work effectively inthe presence of the FBS present in the culture medium.

    In the absence of epidermal separation, the cultures are often contaminated with outgrow-ing keratinocytes, which are readily distinguishable by their cobblestone-like appearance.Contaminating keratinocytes can be removed selectively by incubating 10 to 30 min with0.5% dispase/PBS, because keratinocytes are much more sensitive than fibroblasts to thistreatment. Keratinocytes will be detached, leaving the majority of fibroblasts attached tothe plate.

    35-mm dish

    skin specimen(cut in 2-to 3-mm squares)

    growth medium

    coverslip

    A

    Bcoverslippieces of skin specimen

    Figure 2.1.1 Skin explant culture: (A) top view; (B) side view.

    Current Protocols in Cell Biology

    2.1.3

    Preparation andIsolation of Cells

  • 11. Add 1 ml of 4C trypsin/EDTA solution and incubate 3 to 5 min at room temperaturewhile examining periodically under the inverted phase-contrast microscope. As soonas the fibroblasts round up, add 1 ml ice-cold complete growth medium (DMEM orcomplete RPMI) to inactivate trypsin and harvest cells by gentle pipetting.

    Avoid trypsinization for excessive periods of time and vigorous pipetting, which are the twocommon causes of the low viability problem (see Troubleshooting). Do not pipet the cellsbefore the trypsin has been diluted by addition of complete culture medium.

    Harvest fibroblasts before they become overconfluent and form bundle-like alignments(Fig. 2.1.3B).

    The original skin specimens may be removed using forceps.

    12. Collect fibroblast suspensions into a 15-ml polypropylene centrifuge tube andcentrifuge 10 min at 150 g, 4C. Aspirate the supernatant, tap the pellet to dissociatethe cells, and resuspend them in 100 to 200 l of fresh 4C complete growth medium.

    13. Mix a 10- to 20-l sample of the cell suspension with an equal volume of 0.4% trypanblue/PBS, then count total and viable cells under a microscope using a hemacytome-ter.

    The cell viability as measured by trypan blue exclusion should be >90%.