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 rotation stage vertically, and mount it perpendicularly onto a second, horizontal rotation stage (Figure 1B).</li>
		<li>Mount the horizontal rotation stage onto a two-axis translation stage (Figure 1B).</li>
		<li>Fasten the combination onto a stable surface, such as an optical breadboard.</li>
		<li>Separately mount a rotary tool onto the same breadboard, with the tool positioned in parallel with the breadboard and at approximately the same height as the luer lock connector on the rotation/translation combination stage (Figure 1C).</li>
		<li>Install two cut-off wheels concentrically onto the rotary tool (a smaller, lower grit diamond cut-off wheel over a larger, higher grit stone cut-off wheel) (Figure 1D). 
		NOTE: About a 9 mm difference between the diameters of the two wheels is generally sufficient.</li>
		<li>Position an overhead light source with an adjustable arm over the rotary tool, with the light aimed at the cutting wheels.</li>
		<li>To enhance visualization, position a dissecting microscope over the production setup so that the eyepiece is focused on the cutting discs (Figure 1C). 
		&#8203;NOTE: Alternatively, users can wear magnifying eyewear.</li>
	</ol>
	</li>
	<li>Reshaping the needle tip
	<ol>
		<li>Wear protective eyewear and a surgical mask to prevent fine metal particles from entering the eyes or airways.</li>
		<li>Choose hypodermic needles of the appropriate gauge size, based on experimental requirements.</li>
		<li>Mark off the intended length on each harvesting needle. 
		NOTE: For harvesting from swine skin, 8 mm needles are typically sufficient; although, the length may vary based on experimental needs (e.g., thickness of target skin tissue, needle gauge size). Generally, a 19-gauge needle works well for swine skin.</li>
		<li>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 in step 1.2.3.</li>
		<li>Connect the shortened, blunt needle to the female luer lock connector on the production stage.</li>
		<li>Adjust the horizontal rotation stage so that the needle is at a 12&#176; angle parallel to the cutting discs on the rotary tool (changing the angle will impact the force required for needle insertion).</li>
		<li>Turn on the overhead light and adjust its position while observing the needle under magnification, until the light is reflected off the midline (lengthwise) of the needle.</li>
		<li>Power on the rotary tool, then use the translation stage to advance the needle towards the inner (diamond) cutting disc (Figure 2A).</li>
		<li>Keep advancing the needle slowly against the cutting disc until the cutting disc reaches approximately the midline of the needle (as visualized by the overhead light&#39;s reflection along the midline).</li>
		<li>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 (Figure 2B).</li>
		<li>Retract the needle away from the cutting disc.</li>
		<li>Using the vertical rotation stage, rotate the needle 180&#176;.</li>
		<li>Repeat steps 1.2.9-1.2.10 to reshape the other side of the needle. 
		NOTE: The needle should now have two cutting tips of approximately equal length (Figure&#160;2C and 2D).</li>
		<li>Remove the needle from the production stage.</li>
		<li>Clean the inside bore with a metal wire that is slightly smaller than the needle&#39;s inner diameter.</li>
		<li>Using a sharp wooden stick (e.g., by snapping off the end of the small wooden stick on a cotton tip applicator), remove any burrs that may be still attached to the edges of the newly-formed needle. 
		NOTE: Harvesting needles can be electropolished and sterilized by autoclave if necessary.</li>
	</ol>
	</li>
</ol>
2. Skin Tissue Harvesting
<ol>
	<li>Use the harvesting needles to collect skin microcolumns from ex vivo tissue or live animals.</li>
	<li>For ex vivo skin tissue that is thin (especially from samples where the subcutaneous fat is missing or was trimmed away), hold the target tissue over the opening of a 50 mL centrifuge tube, or stack two pieces of tissue on top of each other, to avoid damaging the needle tips by hitting them against hard surfaces. 
	&#8203;NOTE: For in vivo harvesting from live animals, it is recommended that local lidocaine and epinephrine be administered by intradermal injection for analgesia and to reduce bleeding.</li>
	<li>Assemble the harvesting apparatus according to the amount of microcolumns needed, as described below.</li>
	<li>Low-medium volume option:
	<ol>
		<li>To harvest small to medium amounts of microcolumns, simply fill a standard syringe (10-20 mL syringes usually work well) with normal saline and connect it to a harvesting needle.</li>
		<li>Completely insert the harvesting needle into the donor skin, then retract it.</li>
		<li>Push on the piston of the syringe to flush saline through the harvesting needle and expel the microcolumn that is lodged in the needle bore.</li>
		<li>To speed up the harvesting process, repeat step 2.4.2 3 to 5 times before expelling the microcolumns in step 2.4.3. 
		NOTE: It is usually convenient to expel the microcolumns into a standard cell strainer to ease the subsequent collection of microcolumns.</li>
		<li>If needles become clogged, increase the pressure on the piston to expel the stuck tissue and remove the clog. If simply increasing pressure is insufficient, insert a metal wire through the needle tip opening to clear the clog in the needle bore.</li>
		<li>Keep the microcolumns submerged in saline or medium until use to prevent desiccation . 
		&#8203;NOTE: With an experienced operator, the method described above can be used to harvest microcolumns at a rate of approximately 1 microcolumn per second.</li>
	</ol>
	</li>
	<li>High volume option:
	<ol>
		<li>Create a simple suction-assisted device that can be constructed to facilitate collection of large amounts of microcolumns. 
		NOTE: The device consists of a harvesting needle, 20 mL syringe with luer lock nozzle, suction adapter typically used for liposuction (Figure 3A), and sterile suction canister.</li>
		<li>Remove the plunger from the 20 mL syringe.</li>
		<li>Attach the syringe to a suction adapter.</li>
		<li>Complete the assembly by attaching a harvesting needle to the syringe (Figure 3B).</li>
		<li>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 (which may require connection to a canister port that is marked for outflow rather than inflow).</li>
		<li>Connect the apparatus to a negative pressure source. 
		NOTE: The pressure required depends on needle diameter and length, as previously described8. For the apparatus described in this manuscript, the suction system found in typical operating rooms is generally sufficient.</li>
		<li>Connect a harvesting needle to the syringe.</li>
		<li>Harvest the microcolumns by inserting the harvesting needle into the skin. 
		NOTE: The microcolumns will be drawn into the syringe by suction, then flushed into the suction canister.</li>
		<li>Intermittently dip the harvesting needle into a container of sterile saline during the harvesting procedure to flush the system. 
		NOTE: This saline flush facilitates transport of the microcolumn and ensures they stay hydrated. Alternatively, an MFF luer adaptor can be connected to the luer lock of the syringe, and through a connection tubing, also to a hanging saline drip bag.</li>
		<li>Keep on hand a metal wire, or smaller gauge needle, that is slightly smaller than the harvesting needle&#39;s inner diameter. If the needle becomes clogged, it can be cleared by inserting the metal wire into the needle bore.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>After harvesting, apply a topical antibiotic ointment over the donor sites. 
	NOTE: Additional dressings are generally not required for donor sites.</li>
</ol>

<ol>
	<li>Sun, B. K., Siprashvili, Z., Khavari, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+skin+grafting+and+treatment+of+cutaneous+wounds.">Advances in skin grafting and treatment of cutaneous wounds.</a> Science. 346 (6212), 941-945 (2014).</li><li>Singh, M., 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=Challenging+the+Conventional+Therapy:+Emerging+Skin+Graft+Techniques+for+Wound+Healing.">Challenging the Conventional Therapy: Emerging Skin Graft Techniques for Wound Healing.</a> Plastic and Reconstructive Surgery. 136 (4), 524-530 (2015).</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=Fractional+Skin+Harvesting:+Autologous+Skin+Grafting+without+Donor-site+Morbidity.">Fractional Skin Harvesting: Autologous Skin Grafting without Donor-site Morbidity.</a> Plastic and Reconstructive Surgery. 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. A., 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=Chimeric+autologous/allogeneic+constructs+for+skin+regeneration.">Chimeric autologous/allogeneic constructs for skin regeneration.</a> Military Medicine. 179, 71-78 (2014).</li><li>Ter Horst, B., Chouhan, G., Moiemen, N. S., Grover, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+keratinocyte+delivery+in+burn+wound+care.">Advances in keratinocyte delivery in burn wound care.</a> Advanced Drug Delivery Reviews. 123, 18-32 (2018).</li><li>Wong, V. W., Levi, B., Rajadas, J., Longaker, M. T., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+niches+for+skin+regeneration.">Stem cell niches for skin regeneration.</a> International Journal of Biomaterials. 2012, 926059 (2012).</li><li>Manstein, D., Herron, G. S., Sink, R. K., Tanner, H., Anderson, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractional+photothermolysis:+a+new+concept+for+cutaneous+remodeling+using+microscopic+patterns+of+thermal+injury.">Fractional photothermolysis: a new concept for cutaneous remodeling using microscopic patterns of thermal injury.</a> Lasers in Surgery and Medicine. 34 (5), 426-438 (2004).</li><li>Iriarte, C., Awosika, O., Rengifo-Pardo, M., Ehrlich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+applications+of+microneedling+in+dermatology.">Review of applications of microneedling in dermatology.</a> Clinical, Cosmetic and Investigational Dermatology. 10, 289-298 (2017).</li><li>Anderson, R. 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=Laser+treatment+of+traumatic+scars+with+an+emphasis+on+ablative+fractional+laser+resurfacing:+consensus+report.">Laser treatment of traumatic scars with an emphasis on ablative fractional laser resurfacing: consensus report.</a> Journal of the American Medical Association Dermatology. 150 (2), 187-193 (2014).</li><li>Hogan, S., Velez, M. W., Ibrahim, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneedling:+a+new+approach+for+treating+textural+abnormalities+and+scars.">Microneedling: a new approach for treating textural abnormalities and scars.</a> Seminars in Cutaneous Medicine and Surgery. 36 (4), 155-163 (2017).</li><li>Manuskiatti, W., Fitzpatrick, R. E., Goldman, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+effectiveness+and+side+effects+of+carbon+dioxide+laser+resurfacing+for+photoaged+facial+skin.">Long-term effectiveness and side effects of carbon dioxide laser resurfacing for photoaged facial skin.</a> Journal of the American Academy of Dermatology. 40 (3), 401-411 (1999).</li></ol>

The authors are co-inventors on patents relating to the technology described in this article, from which they have received licensing and royalty revenues through their institution, the Massachusetts General Hospital. J. Tam also consults for Medline Industries, Inc., which holds the commercial license for this technology.

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><tbody><tr><td>Diamond wheel</td><td>Dremel</td><td>545</td><td></td></tr><tr><td>Hypodermic needle (19G)</td><td>Fisher Scientific</td><td>14-840-98</td><td>Other needle sizes could be used, depending on experimental needs</td></tr><tr><td>Stone wheel</td><td>Dremel</td><td>540</td><td></td></tr><tr><td>Syringe (20mL with luer lock)</td><td>Fisher Scientific</td><td>22-124-967</td><td></td></tr><tr><td>Suction adapter</td><td>Tulip Medical</td><td>PA20BD</td><td>Optional, for high volume harvesting</td></tr><tr><td>Suction canister</td><td>Fisher Scientific</td><td>19-898-212</td><td>Optional, for high volume harvesting. Sterilize before use.</td></tr><tr><td>Suction tubing</td><td>Medline</td><td>DYND50216H</td><td>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 ...

apparatus-for-harvesting-tissue-microcolumns

Research

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Bioengineering

5.9K Views.  Massachusetts General Hospital. Deze methode kan helpen bij het beantwoorden van essentiële vragen in het huidweefsel regeneratie veld dat is hoe de verschillende celtypes en structuren die zijn opgenomen in de natuurlijke huid te vervangen. Het belangrijkste voordeel van deze techniek is dat autologe volle dikte huid kan worden verzameld met minimale donor site morbiditeit. Het aantonen van de procedure zal Bill Farinelli zijn, die een onderzoeksmedewerker in ons lab is.Begin deze procedure met het instellen van de...

Video: Apparatus for Harvesting Tissue Microcolumns

Preparing a Microfluidics-Based Vascularized Human Micro-Tumor Model

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

Author Spotlight: Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

Cancer Research

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Immunology and Infection

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Neuroscience

Single Cell Sampling Using Micromanipulation: A Technique to Isolate Single Cells from Cancer Cell Suspension

Source: Chen S. et al. <a href="https://www.jove.com/video/56394" target="_blank">Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization.</a> J. Vis. Exp. (2018)
This video describes the technique for isolating single cells from a pool of target cells to carry out downstream analysis. In this method, a micromanipulator fitted to a microscope is used for high-precision isolation of a single target cell.

Source: Chen S. et al. Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization. J. Vis. Exp. (2018)
This ...

JoVE Encyclopedia of Experiments

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Single Cell Sampling Using Laser Capture Microdissection: A Technique to Harvest Target Cells from a Heterogeneous Cell Population

Source: Chen S. et al. <a href="https://www.jove.com/video/56394" target="_blank">Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization.</a> J. Vis. Exp. (2018)
This video describes the technique for obtaining a single cell from a heterogeneous cell population using laser capture microdissection. The single isolated cell can be used for further genomic studies or cancer research.

Bioscaffold Fabrication by Electrospraying: A Technique to Generate Microcarrier Scaffolds Derived from Extracellular Matrix of Decellularized Tissue

Source: Kornmuller, A. et al. <a href="https://www.jove.com/video/55436" target="_blank">Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms.</a> J. Vis. Exp. (2017)
This video demonstrates the procedure of extracellular matrix-derived microcarrier fabrication by electrospraying. These microcarriers simulate the native tissue-specific microenvironment and are stable in long-term 3D in vitro cell culture models.

Source: Kornmuller, A. et al. Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms. ...

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Assays & techniques

Cryomilling of Decellularized Tissue: A Technique to Obtain Fine Extracellular Matrix Powder

Source: Kornmuller, A. et al. <a href="https://www.jove.com/video/55436" target="_blank">Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms.</a> J. Vis. Exp. (2017)
In this video, we present the protocol to obtain finely ground extracellular matrix powder from lyophilized decellularized tissue by cryomilling. The powdered ECM retains its biochemical composition and can be used for microcarrier synthesis.

An In Vitro Method to Generate Astrocyte Scaffolds within Hydrogel Micro-columns

Source: Katiyar, K. S., et al. <a href="https://app.jove.com/v/55848">Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration.</a> J. Vis. Exp. (2018).
This video demonstrates a method to generate three-dimensional astrocyte scaffolds within hydrogel micro-columns. The inside of the microcolumns are coated with collagen. Astrocytes are introduced, which adhere to collagen and, with further incubation, self-assemble into a three-dimensional scaffold.

1. Development of the Agarose Hydrogel Micro-columns

	Make an agarose 3% weight/volume (w/v) solution by weighing 3 g of agarose and transferring it to a ...

Apparatus for Harvesting Tissue Microcolumns

Samenvatting

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