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

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

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

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

*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

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^TM 150i 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^TM General 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

A. Sample collection of livestock ear tissue

Critical: Ensure that all animal materials are collected in accordance with local guidelines, institutional regulations, and Standard Operating Procedures (SOPs).

1. After the animal has been appropriately euthanized, thoroughly spray the area with 75% ethanol and rub to remove excess dirt and contaminants. Collect the ear by cutting at its base using sterile scissors.

2. Store the ear in a sealed bag or an airtight container at 4 °C or on ice until processing (see General note 1). Wash the ear thoroughly with antibacterial soap and warm water.

3. (ALTERNATIVE) After properly restraining the animal, thoroughly clean the sample area with 75% ethanol and rub to remove excess dirt and contaminants. Using sterile scissors, an ear notch tool, or a biopsy punch, collect 1–2 cm² of skin or ear tissue and store in collection media at 4 °C until processing.

B. Sample processing of tissue

Before you begin: Warm FGM and DPBS wash in a 37–38 °C water bath for at least 30 min.

1. Using sterile scissors, cut a 1–2 cm² section of the ear from the conchal region of the pinna. If possible, avoid the helix and peripheral margins of the external ear (Figure 1).

Note: While fibroblasts can be successfully cultured from nearly any ear location, Figure 1 indicates regions that possess less hair and ear wax contamination, leading to a higher likelihood of success. If samples are collected on live animals, other sampling locations may be necessary. To combat potential contaminants in dirtier locations, incorporate ethanol and antibacterial washes prior to collection.

Figure 1. Goat ear illustration, highlighting the ideal sampling region in green and a good sampling region in yellow

2. Wash the sample once with 75% ethanol and three times with warm water and antibacterial soap. Be sure to rinse the sample with sterile water (or DPBS) between each wash (see General note 2). (Optional) Pat the sample dry with a paper towel to remove excess water.

3. Transport the cleaned sample to a flow hood sterilized using ethanol and UV treatment in a sterile 60 mm Petri dish.

Critical: The following steps should be conducted under a laminar flow hood using aseptic techniques and sterile instruments. A UV light sterilization cycle is also beneficial for reducing contamination. Additionally, a laminar flow hood is not intended to act as or replace a biological safety cabinet but rather provide an aseptic environment for the manipulation and handling of fibroblast cultures. Personnel should wear proper personal protective equipment when needed and use a biological safety cabinet when working with any potentially infectious samples.

(Optional) Add 1–2 mL of DPBS wash to the Petri dish to rinse and moisten the sample during transport to the flow hood. This is especially helpful in longer transportation times to reduce tissue drying.

4. Using a sterile scalpel blade with a handle, shave any hair from the sample. Do not remove any layers of skin. Use tweezers to orient and stabilize the sample (Figure 2). (Optional) Add 1–2 mL of DPBS wash or FGM to keep the sample moist and prevent excessive hair accumulation on the scalpel blade.

Figure 2. Shaving hair on the bovine ear tissue sample in FGM using a scalpel blade with the handle held at a 45° angle

5. Once all hair is removed, rinse the tissue in a new Petri dish with 3–4 mL of DPBS wash. Repeat three times with fresh DPBS wash or until hair remnants are removed (Figure 3).

Note: While the presence of hair does not heavily affect the viability of the culture, it exponentially increases the risk of contamination. Hair fragments trap bacteria and fungus and limit the ability to properly wash the skin sample.

Figure 3. Rinsing the shaved bovine ear sample with DPBS wash

6. Rinse tissue once in 75% ethanol for no more than 30 s. This can be done with a pipette or by briefly submerging the tissue in the ethanol solution. Ensure that both sides of the tissue are rinsed.

7. Rinse tissue in DPBS wash three times. Ensure that both sides of the tissue are rinsed.

8. Coat the tissue with a few drops of betadine. Allow the tissue to rest for 30–60 s. Tap off excess and rinse in DPBS wash twice.

9. Place the cleaned sample in a sterile Petri dish containing a small volume of DPBS wash. Using a new sterile blade, finely mince the tissue into approximately 1 mm² tissue explants (Figure 4). (Optional) Mince tissue in FGM.

Note: The choice between DPBS wash and FGM is largely based on user preference, as neither approach provides a distinct advantage over the other.

Figure 4. Finely mincing the bovine sample in FGM. Use of FGM in place of DPBS wash is optional.

10. Add 2 mL of prewarmed FGM to each well of a 6-well plate. Using sterile tweezers, transfer 5–7 of the minced tissue explants to each well; 1 cm² of tissue is enough for 3–4 wells of a 6-well plate (Figure 5).

Figure 5. Transferring minced bovine tissue to the prepared 6-well plate

11. Incubate the plate at 37.5 °C and 5% CO₂.

C. Establishment of primary fibroblast culture from tissue explants

1. After 24 h of incubation, check the culture plate for signs of contamination such as media color change, putrid scents, or fungal growth. If there are no signs of contamination, allow the culture to incubate for 48–72 h undisturbed. Avoid excessive movement of the plate, as this may dislodge tissue explants and reduce fibroblast outgrowth.

2. After three days of culture, gently add 1 mL of prewarmed FGM to each well. Try not to disturb the tissue explants.

3. Continue to monitor for contamination or infection every 24–48 h until outgrowth is visible. If viable cells are not visible by day 6, remove 1 mL of media from each well and replace with 1 mL of prewarmed FGM. Continue to replace 1 mL of media every 72 h until cells are visible. Cells should initially appear as small circular structures that elongate into spindle-like cells (Figures 6–7) (see General note 3).

Figure 6. Immature fibroblast cells emerging from tissue explants viewed with brightfield illumination. Magnification: 60×.


Figure 7. Elongated fibroblast morphology viewed with brightfield illumination. Magnification: 60×.

4. Forty-eight to seventy-two hours after cells are visible, gently remove all minced tissue with sterile tweezers and discard. Avoid touching the bottom of the culture wells. Smaller tissue explants may be removed with a pipette.

5. Remove all media and gently wash each well twice with 1 mL of warm DPBS wash. Remove all residual wash and replace with 2 mL of prewarmed FGM.

6. Repeat step C5 every 48 h until cells reach at least 60%–70% confluence.

Notes:

1. The ideal cell count to seed a T25 flask is approximately 0.7–1 million cells. At 70% confluence, each 6-well plate has roughly 0.7–0.9 million cells per well, meaning that you may use a 1 well: 1 T25 split ratio.

2. Before you proceed: Prewarm FGM, FBS, 0.05% trypsin-EDTA and DPBS wash in a water bath for at least 30 min.

7. Remove FGM from each well. Gently wash each well twice with 1 mL of DPBS wash. Ensure that all residual wash is removed and discarded.

8. Add 1 mL of 0.05% trypsin-EDTA to each well. Incubate plate at 37.5 °C and 5% CO₂ for 3–5 min until fibroblast cells release from the plate (see General note 4).

Critical: Incubating for longer than 7 min will result in other cell types releasing into your passage and may induce cellular damage to the fibroblasts (see General note 5).

9. Add 0.56 mL of FBS to each well and gently swirl to mix.

Alternative: DPBS or PBS may be used in place of FBS. DPBS is cheaper but can decrease cell viability.

10. Tilting the plate at a 45° angle, use the cell/trypsin mixture in each well to gently rinse excess cells to one side of the well. Transfer the mixture into a 15 mL conical centrifugation tube.

11. Centrifuge at 250× g for 5 min at 20 °C. (Optional) Remove the supernatant and resuspend the cell pellet in 1–4 mL of FGM. Pipette 10 µL of the resuspended cells into a microcentrofuge tube along with 10 µL of 0.4% Trypan Blue solution. Mix well and allow the sample to incubate at 25 °C for 3 min. Load 10 µL of the Trypan Blue/cell suspension onto a hemocytometer slide and analyze with automated cell counter. Viable cells will remain bright, while apoptotic cells will appear blue. Multiply the resulting cell number by the volume of FGM used for the initial resuspension to determine the total estimated cell count.

12. Repeat step C11 to re-pellet the suspended cells or proceed to step C13.

13. Remove the supernatant and resuspend the pellet in 4 mL of warmed FGM. Transfer resuspended cells to a sterile T25 culture flask and incubate at 37.5 °C and 5% CO₂.

D. Maintenance and proliferation of the primary fibroblast cell culture

1. Check culture for contamination, adhesion, and proliferation every 24–48 h. Cells should begin adhering after 4–6 h and elongate after 20 h of passing. Change the FGM every 48–72 h.

2. To refresh FGM, repeat step C5 with modified volumes as shown in Table 1.

Table 1. Volumes for FGM refresh

3. Once cells reach 70%–80% confluence, pass by repeating steps C7–13 with modified volumes and a 1:2 or 1:3 split ratio (Table 2).

Table 2. Volumes for cell passage

E. Cryopreservation of primary fibroblasts

1. To cryo-preserve cells, repeat steps C7–C11, using the volumes from Table 2. Resuspend cells in 1.5 mL of freshly mixed and chilled freezing medium and transfer to a 2 mL cryo vial, aiming for 1–2 million cells per vial. Store at –80 °C for 24–48 h and then move to liquid nitrogen storage tank or other long-term storage.

Validation of protocol

This protocol was used to successfully establish 45 primary fibroblast cell lines (Table 3) over species such as Bos taurus, Bos indicus/taurus cross, Capra hircus (multiple breeds), Ovis aries, and Sus scrofa. These cultures were usually established with ear tissues; however, tissues such as muscle biopsies and fetal skin were also successful in establishing cultures with a 97.8% success rate.

Table 3. Success rates of protocol in different livestock species

Immunocytochemistry staining of one of the adult bovine primary fibroblast cell lines revealed vimentin-positive and cytokeratin-negative marker expression (Figure 8). This is consistent with the expected fibroblast cell markers.

Figure 8. Representative fluorescent immunostaining demonstrating vimentin-positive and cytokeratin-negative staining in bovine fibroblast cells viewed with fluorescence microscopy. Magnification: 100×.

General notes and troubleshooting

General notes

1. Ears can be stored at 4 °C for a maximum of three weeks after collection if properly washed. For the best results, use within two days of collection. Do not use the sample if decomposition odor is present or if hair falls out with gentle washing and/or handling. To reduce tissue damage and cell lysis, do not freeze ear samples.

2. If desired, washing steps may be repeated multiple times to maximize bacterial and fungal removal. However, repeated washing in ethanol can result in tissue damage, so keep ethanol washes to a minimum.

3. Cells and outgrowth are usually visible by day 7 (Table 4). Depending on the age of the animal and storage time before processing, initial growth may take longer. If cells are not visible at 10–14 days, discard and begin again.

4. Goat fibroblasts are often more adherent than other livestock species. You may use 0.1% trypsin-EDTA or incubate for 4–5 min. Do not incubate longer (see General note 5).

5. Prolonged treatment of trypsin-EDTA is toxic to cells. Leaving cells in undiluted trypsin-EDTA for longer than 10 min will cause cellular damage and significantly reduce viability. You may use alternative releasing enzymes or gently tap the plate/flask on a hard surface to release additional cells.

6. In our hands, cultures were routinely used up to passage 15–20; however, the usable passage range may vary by donor, species, tissue source, and culture history. Users should establish an application-specific passage limit using appropriate quality-control assays.

Table 4. Important checkpoints

7. Labeling and line ID examples: Label all cryotubes, flasks, and plates with cell line ID, passage number, date, and researcher initials (Table 5). On cryotubes, be sure to include estimated cell counts. To generate an informative and unique ID, you may use the following example: BF- A3544EF[0125].

Table 5. ID generation for fibroblast cell lines

Troubleshooting

Problem 1: Fungal contamination observed (Figure 9).

Figure 9. Fungal contamination present in fibroblast culture, appearing as feathery structures, viewed with brightfield illumination. Magnification: 60×.

Possible cause: Fungal spores are present on initial samples.

Solution: Discard contaminated samples and cultures and begin again with an increased number of washes. It is recommended to run a sterilization cycle in the incubator before restarting cultures (see General note 2). If fungal contamination is minimal, remove all fungal growths with a pipette and rinse heavily with DPBS wash. Replace with fresh FGM and observe culture closely. Ensure that the incubator and supplies are free of contamination.

Problem 2: Bacterial contamination is observed (Figures 10–11).

Figure 10. Bacterial contamination in fibroblast culture, appearing as sandy debris, viewed with brightfield illumination. Magnification: 60×.


Figure 11. Comparison between healthy fibroblast medium (left) and culture contaminated with bacterial growth (right), indicated by a change in color, turbidity, and strong odor

Possible cause: Bacteria were present on initial samples.

Solution: Discard contaminated samples and cultures and begin again with an increased number of washes. It is recommended to run a sterilization cycle in the incubator before restarting cultures.

Problem 3: Growth is not observed by day 7.

Solution: See General note 3.

Acknowledgments

Funding: This work was supported by the Agriculture and Food Research Initiative’s Animal Breeding, Genetics and Genomics program (A1201) project award nos. 2021-67016-33417 and 2022-08267, from the U.S. Department of Agriculture’s National Institute of Food and Agriculture.

The following figures were created using BioRender: Graphical overview and Figures 1 and 8: https://BioRender.com/y398erv.

Author contributions

Conceptualization, D.H.; Methodology, M.M.; Investigation, M.M.; Writing—Original Draft, M.M.; Writing—Review & Editing, M.M., D.H.; Funding acquisition, D.H.; Supervision, D.H. All authors have read and agreed to the published version of the manuscript.

Competing interests

The authors declare no conflicts of interest.

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