This work was supported by grants from the National Natural Science Foundation of China (grant numbers 81903189 and 82073418) and the Science and Technology Foundation of Guangzhou (grant number 202102020025).

This study was approved by the institutional review board of the Dermatology Hospital, Southern Medical University (2020081). Informed patient consent was obtained before tissues were collectedfrom the individuals.
1. Preparation
NOTE: The following procedures should be performed in a sterile environment under a biological safety cabinet.
<ol>
	<li>Prepare complete culture medium by adding 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-amphotericin B solution (PSA) to high-glucose Dulbecco&#39;s modified Eagle medium (DMEM).</li>
	<li>Prepare phosphate-buffered saline solution (PBS) with PSA by adding 1% PSA into 1x PBS. Prepare PBS with FBS by adding 1% FBS to 1x PBS.</li>
	<li>Prepare several sterilized scissors, forceps, and scalpels by autoclaving.</li>
</ol>
2. Obtaining removed tissues
<ol>
	<li>Obtain tissues from keloid patients through surgery. Collect the fresh keloid tissues in a sterile packing bag or sterile centrifuge tube, and transfer them to a biological safety cabinet in the laboratory as soon as possible. 
	&#8203;NOTE: In this protocol, the size of the acquired keloid tissue was approximately ~20 x 20 x 10 mm3, and the size may vary depending on the surgery.</li>
</ol>
3. Isolation
<ol>
	<li>Take out the keloid tissue using sterile tweezers, and place it in a 50 mL sterile centrifuge tube containing 10-25 mL of PBS with 1% PSA for 10 min. Then, prepare a 6-well plate, and add 4 mL of PBS supplemented with 1% PSA to each well. Take out the tissue using sterilized tweezers, and wash it twice with PBS supplemented with 1% PSA. Using sterile forceps, transfer the tissue sequentially from one well to the next.</li>
	<li>Remove the adipose andepidermis layers using surgical scissors or a surgical scalpel, and leave the dermis layer untouched. Trim and dissect the dermis layer into 3-5 mm2 pieces with scissors, transfer these pieces using sterile forceps to the next well, and wash them in PBS with 1% PSA solution again.</li>
</ol>
4. Culture
<ol>
	<li>Place the dermis tissue pieces in Petri dishes using sterilized forceps; ensure that the number of pieces is between 10 and 30 and the distance between each piece is &#62;5 mm. Put the Petri dishes upside down in a 5% CO2 incubator at 37 &#176;C for 30-60 min until the pieces of tissues dry a little and stick to the Petri dish. Then, add DMEM supplemented with 10% FBS and 1% PSA, and carefully place the Petri dishes into a 5% CO2 incubator at 37 &#176;C. 
	NOTE: Fibroblasts are a morphologically and functionally heterogeneous cell population. Considering this complexity, the isolation and culture steps should be performed with care; select the keloid dermal tissue pieces evenly, and mix these pieces well before placing them in the Petri dish.</li>
	<li>After 3 days, replace half of the supernatant with a complete culture medium. Change the culture medium every 2-3 days. Observe the fibroblasts under a microscope at 40x magnification every day. 
	NOTE: All the steps should be performed gently. Do not move the Petri dish for 2 days after isolating, as the tissue pieces take time to adhere to the Petri dishes.</li>
	<li>When the fibroblasts growing around the tissue pieces reach approximately 90% confluency, remove the tissue pieces and the culture medium. Wash the fibroblasts with sterile 1x PBS, and add 2 mL of sterile 1x trypsin-EDTA solution to the plates.Incubate the cells for approximately ~3-5 min at 37 &#176;C in a humidified 5% CO2 incubator.Gently tap the culture dish, and observe it under a microscope. When the majority of the cells detach from the plate, add 2 mL of complete medium to end the digestion process.</li>
	<li>Transfer the cell suspension to a 15 mL sterile centrifuge tube, and centrifuge the tube at 300 &#215; g for 3 min at room temperature. Discard the supernatant carefully, and resuspend the cell pellet in complete medium.Seed the fibroblasts into a 9 cm cell culture dish, and incubate at 37 &#176;C in a humidified 5% CO2 incubator.</li>
</ol>
5. Maintenance and preservation
<ol>
	<li>Approximately 3-4 days later, when the fibroblasts have grown to 80% confluency, repeat steps 4.2-4.4, and passage the fibroblasts at a ratio of 1:3. Use the passaged fibroblasts for further experiments. 
	&#8203;NOTE: The fibroblast culture should be stopped after 10 passages, because the cells may start to show changed characteristics compared to the original cells.</li>
	<li>Cryopreserve the passaged cells of P1-P3 in liquid nitrogen for further usage.
	<ol>
		<li>Repeat steps 4.2-4.3. Transfer the cell suspension to a 15 mL sterile centrifuge tube, and centrifuge the tube for 3 min at 300 &#215; g. Discard the supernatant carefully, resuspend the cell pellet in 1 mL of cell freezing medium containing 90% FBS and 10% DMSO, and transfer the suspension into cell cryotubes.</li>
		<li>Move the cells into a frozen box, and then put the frozen box in a &#8722;80 &#176;C freezer. After 1 day, transfer the cells to liquid nitrogen for long-term preservation.</li>
	</ol>
	</li>
</ol>
6. Identification of fibroblasts by immunofluorescence staining
<ol>
	<li>Place round coverslips in a 24-well plate, and culture the passaged fibroblasts at a concentration of 1 &#215; 104 cells/well. When the fibroblasts reach 60% confluency, remove the culture medium. Add 1 mL of 4% paraformaldehyde to fix the fibroblasts for 20 min at room temperature, and wash 3x with PBS for 1 min each time. 
	NOTE: Count the number of cells by using a hemocytometer slide under a microscope or an automatic cell counter.</li>
	<li>Remove the PBS, incubate with 0.5% Triton X-100 for 20 min for the permeabilization of cell membranes, and then wash 3x with PBS for 1 min each time. Add PBS with 0.5% bovine serum albumin, and soak for 30 min. Then, remove it, and add the PDGFR-&#945;/vimentin antibody diluted in antibody diluent at 1:1,000. Incubate overnight at 4 &#176;C.</li>
	<li>The next day, remove the primary antibody, wash the cells 3x with PBS for 3 min, and add the secondary antibody Alexa Fluor-555 goat anti-rabbit IgG diluted with antibody diluent at 1:200 to soak for 1 h.</li>
	<li>Remove the secondary antibody, and wash the cells 3x with PBS. Take out the round coverslips using forceps, put them on glass slides, and add 50 &#181;L of 5 &#181;g/mL 4&#39;,6-diamidino-2-phenylindole (DAPI) solution to stain the cellular nuclei. Keep the samples in a wet, dark box, and observe them under a laser confocal fluorescence microscope. 
	&#8203;NOTE: Add a negative control group without a primary antibody but with a secondary antibody to exclude nonspecific staining.</li>
</ol>
7. Identification of fibroblasts by flow cytometry
<ol>
	<li>Collect the cell pellet into a 1.5 mL sterile centrifuge tube, and resuspend with 50 &#181;L of PBS containing 1% FBS. Incubate with anti-CD90 for 30 min in the dark, and add an anti-IgG isotype control to the control group. Then, add 200 &#181;L of PBS containing 1% FBS, and centrifuge the tube for 10 min at 300 &#215; g at 4 &#176;C.</li>
	<li>Remove the supernatant, and add 200 &#181;L of PBS containing 1% FBS. Resuspend and filter the suspension through a cell strainer (70 mesh). Add a 5 &#181;g/mL stock solution of DAPI at 1:100 to the filtrate, and then acquire the results by flow cytometry. Set the forward scatter (FSC) as the abscissa and the side scatter (SSC) as the ordinate, and circle the main cell population. Select the cell population, and set the phycoerythrin as the abscissa and the count as the ordinate for analysis.</li>
</ol>

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There are no conflicts of interest to declare.

Obtaining primary fibroblasts from keloid tissues is a critical basis for further research. Up until now, there have been two methods for acquiring primary fibroblasts: enzyme digestion and explant culture11,12,13,14. However, both traditional methods have limitations, such as susceptibility to contamination, mixing with other types of cells, a long culture period, and a low rate of success<sup...

Keloid, a fibroproliferative disease, manifests as the continuous growth of plaques that often invade the surrounding normal skin without self-limitation and cause various degrees of itching, pain, and cosmetic and psychological burdens for patients1. Fibroblasts, the primary cells involved in keloids, play an essential role in the formation and development of this disease through excessive proliferation, redundant extracellular matrix production, and disorganized collagens2,3. However, the underlying pathogenesis remains unclear, and an effective therapeutic method for keloid is still lacking; therefore, there is an urgent need for further research4,5.
As there is no ideal animal model for keloid research in vivo6,7,building an in vitro model by acquiring primary fibroblasts from keloid tissues can offer feasibility and reliability for keloid research2,6. Primary cells are those derived directly from living tissue, and it is generally recognized that these cells can more closely resemble the physiological state and genetic background of multiple individuals compared with cell lines8,9. Culturing primary cells provides a powerful means to study the growth and metabolism of cells, as well as other cell phenotypes.
At present, there are two methods for acquiring primary fibroblasts: enzyme digestion and explant culture. However, several obstacles have been identified to obtaining primary fibroblasts, such as the risk of contamination by various bacteria or fungi, mixing with other types of cells that are not easily removed, the long period of the culture cycle, the subsequent changes to the cell characteristics compared to the original cells, and so on9. Therefore, developing a feasible and effective process for obtaining primary fibroblasts is the foundation for further studies and applications.This study describes an optimized protocol for extracting primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

<table><tbody><tr><td>1.5 mL sterile centrifuge tube</td><td>JETBIOFIL</td><td>CFT002015</td><td></td></tr><tr><td>15 mL sterile centrifuge tube</td><td>JETBIOFIL</td><td>8076</td><td></td></tr><tr><td>4% polyformaldehyde</td><td>Beyotime Biotechnology</td><td>P0099</td><td>Cell fixation</td></tr><tr><td>50 mL sterile centrifuge tube</td><td>JETBIOFIL</td><td>8081</td><td>Put keloid tissue</td></tr><tr><td>Alexa Fluor-555 goat anti-rabbit IgG&amp;nbsp;</td><td>Abcam</td><td>Alexa&amp;nbsp;Fluor&amp;nbsp;555&amp;nbsp;</td><td>second antibody for immunofluorescence staining assay</td></tr><tr><td>Anti human CD90</td><td>BioLegend</td><td>B301002</td><td>Identify the purity of fibroblasts</td></tr><tr><td>Antibody diluent</td><td>Beyotime Biotechnology</td><td>P0262</td><td></td></tr><tr><td>Biological safety cabinet&amp;nbsp;</td><td>Thermo Scientific</td><td>1300 series A2</td><td>Isolation and culture cells</td></tr><tr><td>Bovine serum albumin</td><td>aladdin</td><td>B265993</td><td>Blocking for immunofluorescence staining assay</td></tr><tr><td>Carbon dioxide incubator</td><td>ESCO</td><td>CCL-170B-8</td><td>Using for culturing cells</td></tr><tr><td>Cell cryotubes</td><td>Corning</td><td>43513</td><td>Store the cells in low temperature</td></tr><tr><td>centrifugal machine</td><td>Thermo Fisher</td><td>ST 16R</td><td>Discard supernatant&amp;nbsp;</td></tr><tr><td>DAPI</td><td>Beyotime Biotechnology</td><td>C1006</td><td>Stain the cellular nucleus</td></tr><tr><td>DMSO</td><td>MP Biomedicals</td><td>196055</td><td>Using for preserving cells</td></tr><tr><td>Dulbecco&#039;s modified eagle medium</td><td>Gibco</td><td>C11995500BT</td><td>Culture medium solution</td></tr><tr><td>Fetal bovine serum</td><td>BI</td><td>04-001-1A</td><td></td></tr><tr><td>Flow cytometer</td><td>BD</td><td>BD FACSCelesta</td><td>Observing the identity of cells</td></tr><tr><td>frozen box</td><td>Thermo Scientific</td><td>&amp;nbsp;5100-0050</td><td></td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>ECLIPSE Ts2</td><td></td></tr><tr><td>Laser confocal microscope</td><td>Nikon</td><td>AIR-HD25</td><td>Observing the immunofluorescence staining assay</td></tr><tr><td>PDGFR-&amp;alpha; antibody</td><td>CST</td><td>3174T</td><td>First antibody for immunofluorescence staining assay</td></tr><tr><td>Penicillin-streptomycin-Am solution</td><td>Solarbio</td><td>P1410</td><td>Add in culture medium solution to avoid contamination</td></tr><tr><td>petri dish</td><td>JETBIOFIL</td><td>7556</td><td>Culture fibroblasts</td></tr><tr><td>Phosphate buffered saline solution</td><td>Gibco</td><td>C10010500BT</td><td>Culture medium solution</td></tr><tr><td>Rabbit (DAIE) mAB IgG XR (R) Isotuge Control (PE)</td><td>Cell Signaling Technology</td><td>5742S</td><td>As a control for flow cytometry</td></tr><tr><td>Round coverslip</td><td>Biosharp</td><td>801007</td><td>Cell culture</td></tr><tr><td>Triton X 100</td><td>Solarbio</td><td>T8200</td><td>Punch holes in the cell membrane</td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25200072</td><td>Used for passaging cells</td></tr><tr><td>Vimentin antibody</td><td>Abcam</td><td>ab8978</td><td>First antibody for immunofluorescence staining assay</td></tr></tbody></table>

isolation, culture, and characterization of primary dermal fibroblasts from human keloid tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFR&#945; and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

The timeline of the protocol is summarized in Figure 1A. Some representative images of the isolation process are shown in Figure 2; the epidermis and adipose layers were carefully removed, and the dermis layer was separated into small fragments of 3-4 mm2, which were inoculated into the Petri dishes.
As shown in Figure 3A, several fibroblast outgrowths of the tissue pieces were observed und...

Watch this Scientific Journal Video about Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue at JoVE.com

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...

isolation-culture-characterization-primary-dermal-fibroblasts-from

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Medicine

2.1K Views.  Southern Medical University. De isolatie en kweek van primaire fibroblasten afgeleid van keloïde weefsel vormen de basis voor verdere studies van keloïden. Fibroblasten uit keloïde weefsel kunnen gemakkelijk worden verkregen via dit protocol, dat een overvloedige en stabiele bron van cellen in het laboratorium kan bieden voor keloïde onderzoek. Begin met het gebruik van een steriel pincet om het keloïde weefsel in een steriele centrifugebuis van 50 milliliter te plaatsen met 10 tot 25 milliliter PBS aangevuld met 1%...

Video: Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

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.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Developmental Biology

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

Cancer Research

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

Genetics

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Bioengineering

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Biology

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

Neuroscience

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells.

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in ...

Human Skin Keratinocyte Isolation: A Method to Culture Primary Keratinocytes from Skin Samples

Johansen, C. et al. <a href="https://www.jove.com/v/56863"> Generation and Culturing of Primary Human Keratinocytes from Adult Skin</a>. J. Vis. Exp. (2017)
This video describes the isolation of keratinocytes&#8212;the most prominent cells of human skin&#39;s epidermal layer, by employing a combination of enzymatic digestion and mechanical disruption methods. This method helps in the preferential growth of keratinocytes over other skin cell types using a selective growth medium.

Johansen, C. et al.  Generation and Culturing of Primary Human Keratinocytes from Adult Skin. J. Vis. Exp. (2017)
This video describes the isolation of ...

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

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