家畜耳组织块原代成纤维细胞培养体系的建立与冻存

Makenna McKendrick; Darren Hagen

doi:10.21769/BioProtoc.5856

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Peer-reviewed

Establishment and Cryopreservation of Primary Fibroblast Cultures From Livestock Ear-Tissue Explants

家畜耳组织块原代成纤维细胞培养体系的建立与冻存

MM Makenna McKendrick

DH Darren Hagen email

发布: 2026年11月05日第16卷第21期 DOI: 10.21769/BioProtoc.5856 浏览次数: 101

评审: Sébastien GillotinTianYeh LimRishith Ravindran

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Abstract

Primary fibroblast cell culture is widely used in livestock research as a renewable genetic source for applications such as cell banking, molecular analyses, and functional studies [1]. Establishing high-quality primary culture requires careful tissue collection, aseptic processing, and optimized cell culture conditions to maximize growth and minimize contamination [2]. This protocol describes a standardized method for establishing primary fibroblast cultures from livestock species, representing multiple breeds, including cattle, swine, sheep, and goats. Although the ear is the primary tissue source described due to its easy accessibility and minimal invasiveness, this protocol may also be used with similar skin or tissue types if needed. Procedures for tissue collection, sample preparation, explant culture, routine maintenance, passaging, and cryopreservation are detailed to support the generation and proliferation of healthy fibroblast cultures suitable for downstream research applications.

Key features

• Describes a reproducible workflow for the establishment, proliferation, passaging, and preservation of fibroblast cultures from livestock species such as goat, pig, sheep, and cow.

• Uses tissue explant culture for gentle, reproducible, and reliable culture establishment.

• Can be used over a variety of breeds, species, animal ages, and tissue types.

Keywords: Livestock (家畜)

Tissue explant (组织外植体)

Primary fibroblasts (原代成纤维细胞)

Cell culture (细胞培养)

Cryopreservation (冻存)

Graphical overview

Background

Fibroblasts are one of the primary connective tissue cells responsible for producing and maintaining the extracellular matrix while contributing to tissue repair and cellular signaling [3,4]. Their ease of culturing and robust growth in in vitro conditions make primary fibroblasts among the most useful cell types for agricultural and biomedical research [3,5,6]. In livestock species, primary fibroblasts serve as valuable resources for genetic conservation [7], cell banking [1], genome editing [8], and other similar areas of research. Because fibroblasts can be established from minimally invasive tissue samples, they provide an effective means of preserving genetic material from animals of diverse breeds, ages, and systems [2]. The ear tissue is particularly well-suited for fibroblast isolation since it is readily accessible, can be preserved for later processing, and does not affect the quality of the carcass. Additionally, ear tissue contains abundant dermal fibroblasts and can be collected from recently deceased carcasses or live animals with minimal stress [2]. Alternatively, fibroblasts may be isolated from a variety of other tissues, including skin biopsies, fetal tissues, tail, and other connective tissues when appropriate [6].

Successful establishment of fibroblast cultures depends on effective sample processing, strict aseptic techniques, appropriate culture conditions, and careful monitoring throughout early culture stages [2]. Standardized protocols improve reproducibility, reduce contamination, and increase the likelihood of obtaining healthy, expandable cell populations suitable for long-term storage and downstream applications. This protocol provides a practical, reproducible approach for establishing primary fibroblast cultures from multiple livestock species and breeds while allowing flexibility for alternative tissue sources.

Materials and reagents

Biological materials

1. Fetal or adult tissue samples (ear, skin, or tissue biopsy)

Reagents

1. Dulbecco’s phosphate-buffered saline without calcium and magnesium (DPBS) (Gibco, catalog number: 14190-144)

2. Dulbecco’s modified Eagle medium (DMEM) (Gibco, catalog number: 11885-084)

3. Fetal bovine serum (FBS) (Gibco, catalog number: 12483-020)

4. Antibiotic-antimycotic 100× (anti-anti 100×) (Gibco, catalog number: 15240-062)

5. 0.05% trypsin-EDTA (Sciencell Research Laboratories, catalog number: NC1672820)

6. Dimethyl sulfoxide Hybri-Max (DMSO) (Millipore Sigma, catalog number: D2650-5X5ML)

7. Skin prep solution Betadine 4oz. bottle 10% strength (Fisher Scientific, catalog number: 503043366)

8. 75% ethanol

9. Antibacterial soap

10. Trypan Blue solution, 0.4% (Gibco, catalog number: 15-250-061)

Solutions

1. Fibroblast growth medium (FGM) (see Recipes)

2. DPBS wash (see Recipes)

3. Freezing media (see Recipes)

4. Collection medium (see Recipes)

Recipes

Before you begin: Aliquot frozen reagents (i.e., FBS, trypsin-EDTA 0.05%, DMSO, anti-anti 100×) into appropriate volumes before storage at -20 °C to minimize multiple freeze/thaw cycles. When possible, use freshly thawed aliquots for each recipe and store mixed solutions in small volumes (indicated on individual recipe instructions) once mixed. Doing so will preserve the integrity of solutions and allow for longer storage and more consistent results.

1. FGM

Reagent	Final concentration	Volume (50 mL)	Volume (250 mL)
DMEM	89% v/v	44.5 mL	222.5 mL
FBS	10% v/v	5 mL	25 mL
Anti-anti 100×	1% v/v (1× final concentration)	500 μL	2.5 mL
Total	n/a	50 mL	250 mL

Mix reagents to the desired volume of FGM under a sterile flow hood, adding anti-anti 100× last. Ensure thorough mixing of each reagent before adding. Filter through a 0.2 μm vacuum filter within 20 min of mixing to remove potential bacterial or fungal contaminants.

Store in 20–50 mL aliquots at 4 °C for up to 30 days. Media should be red in color. Do not use if medium becomes yellow, bright pink, turbid, or putrid.

2. DPBS wash

Reagent	Final concentration	Volume (50 mL)	Volume (250 mL)
DPBS	99% v/v	49.5 mL	247.5 mL
Anti-anti 100×	1% v/v (1× final concentration)	500 μL	2.5 mL
Total	n/a	50 mL	250 mL

Mix reagents to the desired volume of DPBS wash under a sterile flow hood, adding anti-anti 100× last. Ensure thorough mixing of reagents before adding. Optional: For extra precaution, filter through a 0.2 μm vacuum filter to eliminate potential bacterial contaminants.

Store in 20–50 mL aliquots at 4 °C for up to 30 days. Do not use if the wash becomes colored or turbid.

3. Freezing medium

Reagent	Final concentration	Volume (1.5 mL)
DMEM*	80% v/v	1.2 mL
FBS	10% v/v	150 μL
DMSO	10% v/v	150 μL
Total	n/a	1.5 mL

*You may use 90% v/v FBS, 80% v/v PBS, or 80% v/v DPBS in place of 80% v/v DMEM, but ideal results are obtained with 80% v/v DMEM and 10% v/v FBS.

Make fresh for each use. Mix cold reagents under a sterile flow hood to make up 1.5 mL of freezing medium for each cryo vial.

Store at 4 °C or on ice until immediately before use. Do not use the solution if older than 12 h.

4. (ALTERNATIVE 1.A.) Collection medium

Reagent	Final concentration	Volume
DMEM	99% v/v	9.9 mL
Anti-anti 100×	1% v/v	100 μL
Total	n/a	10 mL

Mix reagents to the desired volume of collection medium under a sterile flow hood, adding anti-anti 100× last. Ensure that reagents are thoroughly mixed before adding them.

Store at 4 °C for up to two weeks. For ideal results, use within three days of mixing.

Laboratory supplies

1. 15 mL conical centrifuge tube (Thermofisher Scientific, catalog number: 12565269)

2. 50 mL conical centrifuge tube (Thermofisher Scientific, catalog number: 12565271)

3. Falcon 6-well flat-bottom plates with lids (Corning, catalog number: 21100-002)

4. SureOne aerosol barrier pipette tips, 1,000 µL, filtered (Fisher Scientific, catalog number: 02707404)

5. T25 EasYFlask, TC surface, filter cap, 25 cm²(Thermofisher Scientific, catalog number: 2565351)

6. Cryo vial, internal thread, 2 mL (Corning, catalog number: 1232E26)

7. 60 mm × 15 mm Petri dish (Bellco Glass Corporate, catalog number: 23A00L845)

8. Carbon steel scalpel blades #10 (VWR International, catalog number: 76457-448)

9. Scalpel knife handle No. 3 (VWR International, catalog number: 76457-416)

10. Metal tweezers (United Scientific Supplies Inc., catalog number: S111016), autoclave sterilized

11. Sterile polystyrene serological pipettes, 10 mL (Fisher Scientific, catalog number: 1367610J)

12. 250 mL vacuum filter/storage bottle system, 0.2 μM, nylon (Corning, catalog number: 097612)

13. Scissors, autoclave sterilized

Equipment

1. Heracell^TM150i CO₂ Incubator 150 L (ThermoFisher Scientific, catalog number: 50116047), set to 37.5 °C and 5% CO₂

2. Laminar flow hood, e.g., AirClean 600 PCR Workstation Flow Hood (AirClean Systems, model number: AC632LFUVC)

3. Heated water bath, e.g., Precision^TMGeneral Purpose Water Bath (Fisher Scientific, catalog number: TSFP10PMO, supplier number: TSGP10DUCK), set to 37–38 °C

4. Inverted microscope, e.g., Motic AE31 Elite 30W Trinocular Inverted Microscope (Motic Microscopy, SKU: 1100100200841)

5. (Optional) Automated Cell Counter, e.g., Invitrogen Countess 3 Automated Cell Counter (Fisher Scientific, catalog number: A49891PKG)

Procedure

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文章信息

稿件历史记录

提交日期: Aug 12, 2026

接收日期: Sep 24, 2026

在线发布日期: Oct 26, 2026

出版日期: Nov 5, 2026

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如何引用

McKendrick, M. and Hagen, D. (2026). Establishment and Cryopreservation of Primary Fibroblast Cultures From Livestock Ear-Tissue Explants. Bio-protocol 16(21): e5856. DOI: 10.21769/BioProtoc.5856.