Murine Dermal Fibroblast Isolation by FACS
January 7th, 2016
Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.
The overall goal of this procedure is to isolate fibroblasts using FACS, thereby foregoing in vitro culture, which has been shown to cause a phenotypic change in these cells. This method can help answer key questions in developmental biology and wound healing, such as how to identify fibroblast subtypes involved in scarring and fibrosis. The main advantage of this technique is that in vitro experiments are avoided as adherence to plastic, male to the phenotype of the fibroblast.
Not only can this method provide insights into dermal fibroblasts, but it can also be applied to other systems, such as peritoneal fibroblasts and abdominal adhesions. Begin by shaving and depilating the dorsal skin of the mouse of interest. Then, submerge the denuded mouse in 70%ethanol and place the animal on a clean, sterile surface to dry.
Next, starting at the base of the tail, use forceps to tent the skin and make a transverse cut with dissecting scissors. Then, dissect along the suprafascial plane to harvest the dorsal tissue. When a 60 by 100 millimeter piece of skin has been removed, use the blunt edge of a scalpel to carefully remove any subcutaneous fat.
Then, rinse the harvested skin in betadine, followed by five PBS washes on ice. Using razorblades and dissecting scissors, mince the pieces of dermis in a sterile dish until the samples are uniformly approximately two to three millimeters in size. Then, transfer tissue fragments from up to five mice into the appropriate number of individual 50 milliliter conical tubes containing 20 milliliters of collagenase 4 in dmem.
Agitate the samples vigorously at 37 degrees Celsius. After one hour, transfer the tubes to a sterile hood and pass the tissue flurries through a needless 10 milliliter syringe three to five times. Return the samples to the shaker for another 30 minutes, followed by another three to five pipettes with a 10 milliliter syringe.
Then, filter the samples through a 100 micron strainer into a new 50 milliliter tube and wash the mesh with 20 milliliters of dmem, supplemented with FBS to bring the total volume up to 40 milliliters. Next, spin down the cells and use a sterile glass pipette to aspirate the upper fat layer. When the contaminating adipose sites have been removed, use a new glass pipette to remove the rest of the supernatant and resuspend the pellets in 20 milliliters of dmem plus FBS.
Now, filter the cells through a 75 micron strainer and rinse the mesh with 10 milliliters of dmem plus FBS. Then, spin down the cells again and remove the fat and supernatant as just demonstrated. Resuspend the pellet in 20 milliliters of FACS buffer, setting aside a five milliliter aliquot for the unstained control.
Then, spin down the remaining sample, discard the supernatant and place the pellet on ice. To isolate the fibroblasts by FACS, resuspend the pellets in 500 microliters of lineage antibody incubation mix on ice. After 20 minutes, gently mix five milliliters of FACS buffer, supplemented with DNase with the cells and spin them down.
Wash the pellet in a second five milliliters of DNase. Then, resuspend the cells in 500 microliters of FACS buffer and DNase, reserving a 50 microliter aliquot of cells for the viability dye control. Finally, add viability dye to the remaining sample and sort the cells according to the diagram.
Using a lineage negative depletion approach rather than a positive selection approach avoids pre-selecting for particular subpopulations. Pull transcriptome microarray analysis reveals that cultured fibroblasts isolated by the live harvest and tissue explant methodologies have a high degree of similarity at a transcriptome wide level with a Pearson product-moment correlation coefficient of 0.92. By comparison, cultured fibroblasts differed significantly from a live harvested uncultured fibroblast, establishing the importance of analyzing live harvested fibroblasts over cultured fibroblasts.
After it's development, this technique paved the way for researchers in the field of developmental biology and wound healing to confirm fibroblasts heterogeneity and identify subpopulations responsible for scarring and other forms of fibroses.
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Overview
This article presents a FACS-based protocol for isolating mouse skin fibroblasts without the need for in vitro culture, addressing the challenges posed by fibroblast heterogeneity. The method aims to enhance understanding of fibroblast behavior in vivo, particularly in relation to developmental biology and wound healing.
Key Study Components
Area of Science
- Fibroblast biology
- Developmental biology
- Wound healing
Background
- Fibroblasts play a crucial role in various clinical conditions.
- Traditional research often relies on in vitro methods, which can alter fibroblast phenotype.
- Understanding fibroblast subtypes is essential for addressing scarring and fibrosis.
- In vivo studies provide more accurate insights into fibroblast behavior.
Purpose of Study
- To isolate fibroblasts using FACS without in vitro culture.
- To identify fibroblast subtypes involved in scarring and fibrosis.
- To provide a method applicable to various fibroblast systems.
Methods Used
- Shaving and depilating the dorsal skin of the mouse.
- Submerging the mouse in 70% ethanol for sterilization.
- Using FACS to isolate fibroblasts directly from skin.
- Avoiding in vitro culture to maintain fibroblast phenotype.
Main Results
- The FACS method successfully isolates fibroblasts without altering their phenotype.
- This approach can be applied to other fibroblast types beyond skin.
- Insights gained can inform studies on scarring and fibrosis.
- In vivo isolation enhances the understanding of fibroblast behavior.
Conclusions
- The FACS-based protocol is a valuable tool for fibroblast research.
- It allows for the study of fibroblast behavior in a more physiologically relevant context.
- This method can lead to advancements in understanding and treating fibrosis.
Frequently Asked Questions
Executive Industry Relevance
Isolation of murine dermal fibroblasts by FACS enables direct study of fibroblast heterogeneity and function without the confounding effects of in vitro culture. This approach enhances predictive confidence in early discovery by preserving physiologic transcriptional and proteomic profiles, supporting more accurate target validation and mechanistic de-risking. The method is strategically positioned for portfolio teams seeking to reduce biological ambiguity in fibrosis, wound healing, and stromal biology research.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of fibroblast subtypes implicated in scarring and fibrosis.
- Supports biological de-risking by maintaining native cell states for functional studies.
- Improves predictive confidence in target validation by avoiding culture-induced phenotypic drift.
Screening & Assay Development
- Provides physiologically relevant cell populations for downstream assay development.
- Facilitates reproducible and standardized isolation of heterogeneous fibroblast populations.
- Enables quantitative analysis of fibroblast subpopulations for screening readiness.
Translational & Preclinical Research
- Aligns with disease-relevant models for fibrosis and wound healing studies.
- Supports translational biomarker discovery by preserving in vivo cell characteristics.
- Enables risk-adjusted advancement decisions by clarifying fibroblast-driven pathology.
Pipeline & Workflow Integration
This FACS-based isolation method fits at the interface of early discovery and preclinical research, enabling direct hypothesis testing and pathway clarification in disease-relevant systems.
- Discovery Biology: Supports mechanistic studies of fibroblast heterogeneity and function in vivo.
- Screening: Delivers standardized, reproducible cell populations for assay development and compound evaluation.
- Analytics: Provides quantitative outputs for comparing fibroblast subtypes and conditions.
- Translational Research: Maintains continuity from discovery through preclinical validation in fibrosis and wound healing models.
- Enterprise Reuse: Offers a reusable platform for isolating fibroblasts from multiple tissues and disease contexts.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in fibroblast-driven disease models.
- Operational Value: Standardizes isolation workflows and enhances reproducibility across studies.
- Strategic Value: Enables better go/no-go decisions by providing physiologically relevant data early in the pipeline.
- Portfolio Impact: Supports risk-adjusted prioritization of fibrosis and wound healing programs.
Implementation Considerations
- Requires expertise in FACS operation and cell surface marker selection.
- Demands access to advanced flow cytometry instrumentation and analytical infrastructure.
- Necessitates cross-team standardization of gating strategies and controls.
- Adaptable to other tissue sources with appropriate protocol modifications.
- Limited by the need for fresh tissue and potential variability in cell yield.
