Name: Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts (Video) | JoVE Journal
Uploaded: 2013-07-07T13:00:00.000+00:00
Duration: 6 min 19 s
Description: Learn skin punch biopsy explant culture to derive primary human fibroblasts for cell banking and regenerative medicine research. This protocol yields 15-20 million cells at low passage within 4-8 weeks.

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5 minutes

Development of this protocol was funded by the California Institute for Regenerative Medicine (CIRM, TR1-01246) and Parkinson Alliance.

The skin punch biopsy obtained using standard procedure3 (e.g. obtained with 4mm round Visipunch instrument) should be kept in complete DMEM 20% FBS media on ice. Once the sample has arrived in the laboratory, process the biopsy as soon as possible.
1. Preparation of the Skin Punch Biopsy
STEPS 1.1- 1.3 ARE TO BE PERFORMED INSIDE A BIOSAFETY CABINET
<ol>
 <li>Prepare in advance: Add 1 ml of 0.1% Gelatin to each well of a 6-well plate. Set the plate aside for 30-60 min. Aspirate the gelatin solution and add 800 &mu;l of complete DMEM/20% FBS media to each well. Ensure that the entire surface of the well is covered with media. </li>
 <li>Invert the lid of a sterile 10 cm tissue culture dish and add 1.5 ml of DMEM/ 20% FBS media to the middle of the lid and spread out the media drop with the tip of the serological pipette. </li>
 <li>Using a sterile forceps, place the skin biopsy piece in the media on the dish. </li>
</ol>
2. Dissection of the Skin Punch Biopsy
STEPS 9 2.1-2.2 ARE TO BE PERFORMED INSIDE A HORIZONTAL LAMINAR FLOW HOOD
<ol>
 <li>Place the bottom portion of 10 cm dish on inverted lid, and transfer the skin biopsy to the dissecting microscope in the laminar flow hood. </li>
 <li>Dissect a 4-mm round skin biopsy into 12-15 evenly sized pieces with sharp edges by cutting pieces in equal halves using one scalpel to hold the biopsy in place and the second scalpel to cut with a rolling motion in one direction. Pieces with ragged edges contribute to poor attachment/cell outgrowth. </li>
</ol>
3. Transfer of Dissected Skin Biopsy Pieces into Tissue Culture Plates 
STEPS 3.1-3.6 ARE TO BE PERFORMED INSIDE A BIOSAFETY CABINET
<ol>
 <li>Place the bottom portion of the 10 cm tissue culture dish on top of the inverted lid, and transfer dish back into to the biosafety cabinet.</li>
 <li>Using a pointed forceps, place 2-3 biopsy pieces into each well of the prepared 6-well plate containing 800 &mu;l and not on dry wells. Use tapping or sliding motion to get the pieces to attach to the bottom of the well. A scalpel is useful to remove any biopsy pieces from the forceps. </li>
 <li>Place the 6-well plate in the 37 &deg;C incubator. Monitor daily to ensure there is a film of media coating the bottom of the well for the first week; add ~200 &mu;l every 2 days to replace any evaporated media.</li>
 <li>After one week, increase amount of media to 2 ml of complete DMEM/20%FBS and change media every 2-3 days. </li>
 <li>Once fibroblasts are confluent in each well to the point where the fibroblasts are reaching the edges of the well, trypsinize and passage 6-well plate into 2X T75 flasks (passage 1). The tissue pieces can be transferred as well. They will not attach and be washed out during the next media change. </li>
 <li>Once fibroblasts are confluent, transfer them to 3X T175 flasks (passage 2), freeze in complete DMEM media plus 10% DMSO at 1x106 cells/ml per vial. </li>
</ol>
4. Characterization the Fibroblasts through Immunostaining
<ol>
 <li>Culture fibroblasts in 8 well chamber slide coated with gelatin.</li>
 <li>When cells are at 80% confluency, aspirate the medium from each well.</li>
 <li>Fix cells in 4% Paraformaldehyde for ten minutes at room temperature. </li>
 <li> Wash the wells 3 times with 1X PBS.</li>
 <li>Permeabilize the cells with 150 &mu;l of 0.3% Triton X-100 (in PBS) for 5 min at room temperature. </li>
 <li>Wash the wells 3 times with 1X PBS.</li>
 <li>Add 200 &mu;l blocking solution (PBS + 5% Normal Goat Serum (Vector, S-1000)) for 1 hr at room temperature.</li>
 <li>Prepare a 1:250 dilution of Anti-SERPHIN-1 (Anti-rabbit, mAB, Sigma, S5950-200 &mu;l) in blocking solution.</li>
 <li>Aspirate the blocking solution, and add 150 &mu;l of the 1:250 dilution of AntiSERPINH-1. Incubate at 1-2 hr at room temperature, or at 4 &deg;C overnight. </li>
 <li>Wash the wells 3 times with 1X PBS.</li>
 <li>Prepare a 1:200 goat-anti-rabbit monoclonal antibody (Alexa Fluor Goat anti-rabbit IgG(H+L), Invitrogen, A11008) in blocking solution. </li>
 <li>Add 150 &mu;l of 1:200 goat-anti-rabbit monoclonal antibody to each well. Incubate for 1 hr, in dark, at room temperature. </li>
 <li>Wash wells once with 1xPBS. </li>
 <li>Aspirate the PBS, and carefully lift the chambers off to reveal just the slide.</li>
 <li>Mount the slide with a 2-3 drops of mounting medium containing DAPI (Vector, H-1200).</li>
 <li>Analyze the slide under a fluorescent microscope. </li>
</ol>

<ol><li>Nguyen, H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LRRK2+mutant+iPSC-derived+DA+neurons+demonstrate+increased+susceptibility+to+oxidative+stress%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress.</a> Cell Stem Cell. 8, 267-280 (2011).</li>
<li>Mak, S. K., Tewari, D., Tetrud, J. W., Langston, J. W., Schule, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+dysfunction+in+skin+fibroblasts+from+a+Parkinson%27s+disease+patient+with+an+alpha-synuclein+triplication%5BTitle%5D)+AND+%22Journal+of+Parkinson%27s+Disease%22%5BJournal%5D)">Mitochondrial dysfunction in skin fibroblasts from a Parkinson's disease patient with an alpha-synuclein triplication.</a> Journal of Parkinson's Disease. 1, 175-183 (2011).</li>
<li>Punch Zuber, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((biopsy+of+the+skin%5BTitle%5D)+AND+%22Am.+Fam.+Physician%22%5BJournal%5D)">biopsy of the skin.</a> Am. Fam. Physician. 65, 1155-1164 (2002).</li>
<li>Takashima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+of+fibroblast+cultures%5BTitle%5D)+AND+%22Curr.+Protoc.+Cell+Biol%22%5BJournal%5D)">Establishment of fibroblast cultures.</a> Curr. Protoc. Cell Biol. Chapter 2, Unit 2, 1(2001).</li>
<li>Kuroda, K., Tajima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HSP47+is+a+useful+marker+for+skin+fibroblasts+in+formalin-fixed%2C+paraffin+embedded+tissue+specimens%5BTitle%5D)+AND+%22J.+Cutan.+Pathol%22%5BJournal%5D)">HSP47 is a useful marker for skin fibroblasts in formalin-fixed, paraffin embedded tissue specimens.</a> J. Cutan. Pathol. , 241-246 (2004).</li>
</ol>

The authors have nothing to disclose.

With this protocol, relatively pure cultures of skin fibroblasts can be obtained. The fibroblasts have characteristic morphological features of elongated, spindle-like cell bodies, round to oval cell nuclei, and the fibroblasts grow aligned and in bundles when confluent. The media supports the growth of fibroblasts whereas other cell populations e.g. keratinocytes need additional supplements and growth factors, or are mitotically less active, this allows for relatively pure fibroblast cultures. 
<p class="...

<table><tbody><tr><td>Dissecting microscope</td><td>Leica</td><td>S6E</td><td></td></tr><tr><td>10 cm Tissue Culture Petri dish</td><td>VWR</td><td>25382-166</td><td></td></tr><tr><td>6-well Tissue Culture plate </td><td>VWR</td><td>73520-906</td><td></td></tr><tr><td>Stainless steel disposable sterile scalpels (2), blade No#15, Miltex</td><td>VWR</td><td>21909-660</td><td></td></tr><tr><td>Sterile pointed-tip forceps</td><td></td><td></td><td></td></tr><tr><td>50 mL Conical tubes</td><td>VWR</td><td>21008-940</td><td></td></tr><tr><td>2 mL Serological Pipettes</td><td>VWR</td><td>89130-894</td><td></td></tr><tr><td>5 mL Serological Pipettes</td><td>VWR</td><td>89130-896</td><td></td></tr><tr><td>10 mL Serological Pipettes</td><td>VWR</td><td>89130-898</td><td></td></tr><tr><td>Pasteur Pipettes</td><td>VWR</td><td>14672-380</td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;Table A. Table of specific reagents and equipments.&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td>1X DMEM: High Glucose</td><td>Invitrogen/Gibco</td><td>11960-069</td><td></td></tr><tr><td>20% Fetal Bovine Serum</td><td>Invitrogen/Gibco</td><td>26140-079</td><td></td></tr><tr><td>1X L-Glutamine</td><td>Invitrogen/Gibco</td><td>25030-164</td><td></td></tr><tr><td>1X MEM Non-essential Amino Acids </td><td>Invitrogen/Gibco</td><td>11140-076</td><td></td></tr><tr><td>1X Penicillin/Streptomycin</td><td>Invitrogen/Gibco</td><td>15140-163</td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;&lt;strong&gt;Table B. Reagents.&lt;/strong&gt; &lt;/p&gt;</td></tr></tbody></table>

skin punch biopsy explant culture for derivation of primary human fibroblasts

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.

Keratinocytes growing out of the biopsy pieces as soon as 48 hr after dissection. First fibroblasts outgrowth can be observed about one week after processing. Once the wells have reached confluency, fibroblasts are passaged for two more passages to reach three 150 cm flasks (T150) and cells are cryopreserved. We generate with this method 15-20 million cells. The fibroblasts are positive for anti-SERPINH1 (also known as HSP-47) (Figure 1), which is a collagen-specific molecular chaperone localized in the...

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 ...

Hello.Welcome to the skin Biopsy Processing video tutorial. Start by placing the required Materials in the tissue culture hood. All objects entering the hood must be sprayed with 70%ethanol and wiped down, labeled a plate with the PI number, the date, initials, and the type of media at one milliliter of 0.1%gelatin to each wall, swirl the plate around to ensure that the bottom is coated with gelatin.Let the Plate sit for an hour before use. The first step is to transfer the biopsy from the tube to the cutting dish. A cutting dish is a lid of a 10 centimeter Petri plate on which the biopsy will be dissected.Add 1.5 milliliter of DMEM 20%FBS to the center of The cutting dish. Spread the media out from the center. Here you can see the skin biopsy.The next step is to Transfer the biopsy from the tube into the center of the cutting dish. Using sterile forceps, place the skin biopsy piece at the center. Be careful while doing this, it is essential to maintain sterility.Cover the cutting dish and transfer it to the dissecting microscope in the laminar flow hood. The next step is to dissect the biopsy into 12 to 18 evenly sized Pieces. Handle the scalpels with care.When you're ready, uncover the biopsy. Use one scalpel to pin down the biopsy. Move the second scalpel up and down in a one direction slicing motion.Cut the biopsy in half. Continue cutting each piece in half until you have 12 to 18 pieces. The ideal Size of each biopsy piece takes up approximately 70 to 80%of the micrometer in the left ocular objective of the inverted microscope.Avoid pieces smaller than 50%of the micrometer Count. To Ensure there are 12 to 18 pieces. The next step will be done inside the tissue culture hood.Cover the cutting dish and transfer it back into the tissue. Culture hood aspirate gelatin from the six well plate add 700 microliters of DMEM 20%FBS to each. Well, it is a good Practice to add Media against the sidewall of the container.Make sure the well is covered with a thin film of media. Using forceps plays two to three biopsy pieces into each wall of the six. Well plate.Use a tapping or sliding motion to get the pieces to attach to the bottom of the wall. A scalpel can be used to remove any stuck biopsy pieces from the forceps. The cells that will grow on this plate will be at passage zero.This is how the plate should look at the end. Here's one of the pieces under an inverted microscope. This is the skin biopsy on day one.Usually you see attachment within one to three days. Keratinocytes start appearing within a week's time. Fibroblasts can be seen within two weeks.Sometimes the keratinocytes are not observed. Usually it takes three weeks for the fibroblasts to become compliment and ready for packaging.

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Research

JoVE Journal

Medicine

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Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

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.

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Learn skin punch biopsy explant culture to derive primary human fibroblasts for cell banking and regenerative medicine research. This protocol yields 15-20 million cells at low passage within 4-8 weeks.

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

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