This work was supported in part by the Army, Navy, NIH, Air Force, VA and Health Affairs to support the AFIRM II effort, under Award No. W81XWH-13-2-0054. The U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick, MD 21702-5014 is the awarding and administering acquisition office. Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the Department of Defense.

All work involving live animals and animal tissue samples have been approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee (IACUC).
1. Production of Harvesting Needles
<ol>
	<li>Setup of the production stage
	<ol>
		<li>Secure a female luer lock connector onto a post, and mount the post onto a rotation stage so that the luer lock is at the center of the stage (Figure 1A).</li>
		<li>Position this first rotatio...

<ol>
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Global Open. 1 (6), 47 (2013).</li><li>Tam, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+full-thickness+skin+by+microcolumn+grafting.">Reconstitution of full-thickness skin by microcolumn grafting.</a> Journal of Tissue Engineering and Regenerative Medicine. 11 (10), 2796-2805 (2017).</li><li>Huang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+hair+and+other+skin+appendages:+A+microenvironment-centric+view.">Regeneration of hair and other skin appendages: A microenvironment-centric view.</a> Wound Repair and Regeneration. 24 (5), 759-766 (2016).</li><li>Fernandes, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-mechanical+fractional+skin+rejuvenation.">Micro-mechanical fractional skin rejuvenation.</a> Plastic and Reconstructive Surgery. 131 (2), 216-223 (2013).</li><li>Rettinger, C. L., Fletcher, J. L., Carlsson, A. H., Chan, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+epithelialization+and+improved+wound+healing+metrics+in+porcine+full-thickness+wounds+transplanted+with+full-thickness+skin+micrografts.">Accelerated epithelialization and improved wound healing metrics in porcine full-thickness wounds transplanted with full-thickness skin micrografts.</a> Wound Repair and Regeneration. 25 (5), 816-827 (2017).</li><li>Franco, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractional+skin+harvesting:+device+operational+principles+and+deployment+evaluation.">Fractional skin harvesting: device operational principles and deployment evaluation.</a> Journal of Medical Devices. 8 (4), 041005 (2014).</li><li>Rasmussen, C. 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The methods described here are intended to enable the collection of tissue microcolumns in sufficient quantities for in vivo large animal studies, using tools made from commercially available laboratory supplies. This apparatus has been used previously in harvesting tissue from excised human skin4,9 as well as live swine skin3. The specific parameters described are those that were found to be most suited for use in swine. It is ex...

Autologous skin grafting is the mainstay of wound repair, but it is limited by donor site scarcity and morbidity, leading to concerted efforts in recent decades to develop new therapeutic options to replace conventional skin grafting1,2. We recently developed an alternative method of harvesting skin to harness the benefits of full-thickness skin grafting while minimizing donor site morbidity. By collecting full-thickness skin in the form of small (~0.5 mm diameter) &#34;microcolumns&#34;, donor sites are able to heal rapidly and without scarring under normal circumstances (for potential exceptions, see the discussion section below)3. Microcolumns can be applied directly into wound beds to accelerate wound closure, reduce contraction3, and restore a diverse range of epidermal and dermal cell types and functional adnexal structures4, many of which are lacking in conventional split-thickness skin grafting or current bioengineered skin substitutes5. The ability of microcolumns to augment healing and of their donor sites to heal without scarring have both been independently validated by other research groups6,7.
We have previously developed a laboratory harvesting system to enable the collection of microcolumns at scale8; however, this system is composed of many customized components that are not widely available. Here, we describe in detail the process for producing harvesting needles, as well as simple collection systems, made from mostly off-the-shelf components, that can be used to achieve high-volume harvesting. The apparatus described in this manuscript is suitable for in vitro and animal work, but not for use in humans. A clinical device with FDA clearance for applying this technique in humans is commercially available but will not be discussed in detail here.

<table border="0" cellpadding="0" cellspacing="0" style="width:688px;" width="687">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Diamond wheel</td>
			<td style="width:135px;">Dremel</td>
			<td style="width:119px;">545</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:216px;">Hypodermic needle (19G)</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">14-840-98</td>
			<td style="width:219px;">Other needle sizes could be used, depending on experimental needs</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Stome wheel</td>
			<td style="width:135px;">Dremel</td>
			<td style="width:119px;">540</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Syringe (20mL with luer lock)</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">22-124-967</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Suction adapter</td>
			<td style="width:135px;">Tulip Medical</td>
			<td style="width:119px;">PA20BD</td>
			<td style="width:219px;">Optional, for high volume harvesting</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:71px;width:216px;">Suction canister</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">19-898-212</td>
			<td style="width:219px;">Optional, for high volume harvesting. Sterilize before use.</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:59px;width:216px;">Suction tubing</td>
			<td style="width:135px;">Medline</td>
			<td style="width:119px;">DYND50216H</td>
			<td style="width:219px;">Optional, for high volume harvesting</td>
		</tr>
	</tbody>
</table>

apparatus for harvesting tissue microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns of full-thickness skin tissue. The small size of the microcolumns allows donor sites to heal quickly without causing donor site scarring, while harvesting full-thickness tissue enables the incorporation of all cellular and extracellular components of skin tissue, including those associated with deeper dermal regions and the adnexal skin structures, which have yet to be successfully reproduced using conventional tissue engineering techniques. The microcolumns can be applied directly into skin wounds to augment healing, or they can be used as the autologous cell/tissue source for other tissue engineering approaches. The harvesting needles are made by modifying standard hypodermic needles, and they can be used alone for harvesting small amounts of tissue or coupled with a simple suction-based collection system (also made from commonly available laboratory supplies) for high-volume harvesting to facilitate studies in large animal models.

The harvesting needles should be able to collect microcolumns of full-thickness skin tissue with approximately a 80-90% success rate, and each microcolumn should contain epidermis, dermis, and some subcutaneous fat (Figure 4). If the success rate of harvesting is low, or if it becomes difficult to insert a needle into tissue, then a new needle is likely needed. If the success rate for harvesting is consistently low, even with new needles, then the needles are...

Watch this Scientific Journal Video about Apparatus for Harvesting Tissue Microcolumns at JoVE.com

Apparatus for Harvesting Tissue Microcolumns

Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting.

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns ...

This method can help answer essential questions in the skin tissue regeneration field which is how to replace the diverse cell types and structures that are contained in natural skin. The main advantage of this technique is that autologous full-thickness skin can be collected with minimal donor site morbidity. Demonstrating the procedure will be Bill Farinelli, who is a research associate in our lab.Begin this procedure with setup of the production stage as described in the text protocol. Install two cutoff wheels concentrically onto the rotary tool. Use a smaller lower grid diamond cutoff wheel over a larger higher grid stone cutoff wheel.Position an overhead light source with an adjustable arm over the rotary tool with the light aimed at the cutting wheels. To enhance visualization, position a dissecting microscope over the production setup so that the eyepiece is focused on the cutting discs. When reshaping the needle tip, wear protective eyewear and a surgical mask to prevent fine metal particles from entering the eyes or airways.Choose hypodermic needles of the appropriate gauge size based on experimental requirements. Mark off the intended length on each harvesting needle. Lower the needle perpendicularly to the rotary tool with the power on using the edge of the outer cutting disc to cut off the excess length of the needle at the point marked.Connect the shortened blunt needle to the female Luer lock connector on the production stage. Adjust the horizontal rotation stage so that the needle is at a 12 degree angle parallel to the cutting discs on the rotary tool. Turn on the overhead light and adjust its position while observing the needle under magnification until the light is reflected off the midline of the needle.Power on the rotary tool. Then use the translation stage to advance the needle toward the inner cutting disc. Keep advancing the needle slowly against the cutting disc until the cutting disc reaches approximately the midline of the needle.Slowly move the cut needle surface from the inner diamond wheel onto the outer stone wheel to finish the cut needle surface with a finer polish. Retract the needle away from the cutting disc. Using the vertical rotation stage, rotate the needle 180 degrees.Repeat these steps to reshape the other side of the needle. Now remove the needle from the production stage. Clean the inside bore with a metal wire that is slightly smaller than the needle's inner diameter.Using a sharp wooden stick, remove any burrs that may still be attached to the edges of the newly formed needle. When a high volume of microcolumns is needed, remove the plunger from a 20 milliliter syringe. Then attach the syringe to a suction adapter.Complete the assembly by attaching a harvesting needle to the syringe. Use a piece of sterile suction tubing to connect the suction adapter to a sterile suction canister. Make sure the harvesting apparatus is connected to a canister input that allows fluid to flow into the canister unimpeded.Now connect the apparatus to a negative pressure source. Harvest the microcolumns by inserting the harvesting needle into the skin. Intermittently dip the harvesting needle into a container of sterile saline during the harvesting procedure to flush the system.Keep on hand a metal wire or a needle that is slightly smaller than the harvesting needle's inner diameter. If the needle becomes clogged, it can be cleared by inserting the metal wire into the needle bore. When the desired amount of microcolumns have accumulated in the canister, disconnect the device from suction.Then pour the contents of the suction canister out through a filter to collect the microcolumns. Shown here is a finished harvesting needle viewed from the front and from the side. Note the two cutting points at the tip.The harvesting needles should be able to collect microcolumns from full-thickness skin tissue with an approximate 80 to 90%success rate. In this figure, check marks represent one millimeter. Each microcolumn should contain epidermis, dermis, and some subcutaneous fat.Microcolumns can be applied directly to wound healing or they may be combined with different matrix materials to produce combination constructs. Microcolumns can also be maintained in culture for in vitro studies. While performing high-volume harvesting, it's important to remember to incorporate an intermittent saline flush.Though this method was designed for skin wound repair, a similar principle could be applied to other organ systems such as muscles where autologous tissue grafting is needed.

apparatus-for-harvesting-tissue-microcolumns

Research

JoVE Journal

Bioengineering

5.9K visualizações.  Massachusetts General Hospital. Este método pode ajudar a responder perguntas essenciais no campo de regeneração do tecido da pele, que é como substituir os diversos tipos de células e estruturas que estão contidas na pele natural. A principal vantagem dessa técnica é que a pele autóloga de espessura total pode ser coletada com morbidade mínima do local do doador. Demonstrando o procedimento estará Bill Farinelli, que é um pesquisador associado em nosso laboratório.Inicie este procedimento com a configur...