Name: tissue characterization after a new disaggregation method for skin micro-grafts generation
Uploaded: 2016-03-04T13:00:00.000+00:00
Duration: 9 min 30 s
Description: Watch this Scientific Journal Video about Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation at JoVE.com

The authors wish to thanks Dr. Federica Zanzottera for contributing to the study performing flow cytometry analysis.

Ethics statement: since the clinical application of the protocol requires the use of cutaneous autologous tissue of the patient, its characterization in vitro was performed before clinical use on homologous cutaneous tissue at Emilia Romagna Regional Skin Bank following the guidelines of National Rules on harvesting, processing and distributing tissues for transplantation (CNT 2013).
1. Bio-complex Building for Clinical Application
NOTE: This protocol is clinically based on the use of Rigeneracons (tissue disruptor) and the Rigenera Machine (tissue disruptor system) (Figure 1A). The tissue disruptor is a biological medical disruptor of human tissues able to disrupt small pieces of tissues using a grid provided by hexagonal blades and filtering cells and components of extracellular matrix with a cut-off of about 50 microns.
<ol>
	<li>Collect skinsamples of the patient through a biopsy punch (Figure 1B) and disaggregate adding 1 ml of saline solution for each piece to obtain autologous micro-grafts 6,7,9 (or see step 2.1).</li>
	<li>Place 1 ml of micro-grafts on collagen sponges (Figure 1C) toform bio-complexes to use for clinical application.</li>
	<li>Culture another 1 ml of micro-grafts on collagen sponges in the presence of 6 ml of DMEM medium supplemented with 10% Fetal Bovine Serum at 37 &#176;C in a 5% CO2 humidified atmosphere.</li>
	<li>Following 3 days of culture, fix the bio-complexes with 0.3% Paraformaldehyde for 10 min at RT. Pour the paraffin with a specific dispenser directly onto the sample. Obtain slices with a microtome with a thickness of 5 &#181;m and put directly in a glass-slide.</li>
	<li>Immerse paraffin slices of 5 &#181;m in a glass for histological analyses, containing 15 - 20 ml Xylene (commercially available - a mix of m-xylene (40 - 65%), p-xylene (20%), o-xylene (20%) and ethyl benzene (6 - 20%) and traces of toluene, trimethyl benzene, phenol, thiophene, pyridine and hydrogen sulfide) for 3 min each.</li>
	<li>Immerse the slices in decreasing grades (100% to 70%) of ethanol (100% ethanol for 1hr, 95% ethanol for 1 hr, 80 % ethanol for 1hr, 70% ethanol for 1 hr) and then deionized water for de-paraffinizing and rehydrating the sections.</li>
	<li>Stain the sections with 1 - 2 ml of 1g/L Ematoxylin for 1 - 2 min and subsequently rinse in water to remove any Ematoxylin surplus.</li>
	<li>Stain the sections for 4 - 5 min with Eosin Y alcoholic solution at 1% of concentration mixed with ethanol 70% and diluted in water.</li>
	<li>Use 1 - 2 ml of Eosin Y for each slide section and rinse under running tap water.</li>
	<li>Immerse sections in increasing grades of ethanol (see step 1.6) and finally, after a passage in Xylene for 1h, coverslip with a based-mounting mediumand observe under a light microscope at 100X magnification (Figure 1D).</li>
</ol>
2. Collection, Disaggregation and In Vitro Analysis of the Tissues
<ol>
	<li>Using a dermatome, take independent skin tissue, papillary de-epidermized dermis (Ded) or reticular dermis (Dermis) respectively of 0.6 mm, 1 mm or 2 mm in thickness from the trunk area of 4 different multi-organ and/or multi-tissue donors in a range from 40 to 55 years, following National Rules on harvesting, processing and distributing tissues for transplantation (CNT 2013).
	<ol>
		<li>Gently rinse all samples in 0.9% NaCl solution putting them in a dish on an orbital shaker for 5 min.</li>
		<li>Using the 5-mm biopsy punch, create samples which are uniform in diameter from the skin tissue, Ded and Dermis and weigh all tissue specimens before the disaggregation.</li>
		<li>Insert eight, three or four uniform samples of skin tissue, Ded or Dermis respectively, in the tissue disruptor, adding 1.5 ml of saline solution for the disaggregation.</li>
		<li>Perform different times of disaggregation for all tissue samples as indicated in Table 1.</li>
		<li>Use a correspondent number of punch biopsies derived from intact tissue samples as controls.</li>
		<li>After mechanical disaggregation, aspirate the saline solution containing micro-grafts and place separately each sample in a single well of a 12-well plate. Perform the same protocol for intact control punch biopsies.</li>
		<li>Add 1ml of RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics to each sample.</li>
		<li>Evaluate the cell viability immediately. To each well containing a micro-graft (obtained by the simultaneous disaggregation of eight, three or four uniform samples of skin tissue, Ded or Dermis respectively) add 1ml of medium containing 0.5 mg/ml of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) solution and incubate for 3 hr at 37 &#176;C in an atmosphere of 5% CO2/air. Perform the same protocol for intact control punch biopsies.</li>
		<li>After incubation, remove all medium containing MTT and add to each sample 1 ml dimethyl sulfoxide (DMSO) for 10 min.</li>
		<li>Transfer each sample and DMSO in a cuvette and read at optical density (OD) at 570 nm using a spectrophotometer. Calculate cell viability as the ratio of absorbance at 570 nm and the weight in grams (gr) of tissue used before disaggregation. Perform the same protocol for intact control punch biopsies.</li>
	</ol>
	</li>
	<li>After mechanical disaggregation, aspirate the saline solution containing micro-graft derived from skin tissue, Ded and Dermis samples of a single donor.
	<ol>
		<li>Place separately each sample in a single well of a 12-well plate or in a culture flask for cell viability test and morphological analysis respectively.</li>
		<li>Culture micro-grafts adding 1 ml (12-well plate) or 5 ml (culture flask) of RPMI 1640 medium supplemented with 10% FBS and antibiotics at 37 &#176;C in an atmosphere of 5% CO2/air for 24 hr&#160;or 7 days.</li>
		<li>Evaluate the cell viability after 24 hr or 7 days (repeat steps 2.1.8-2.1.10).</li>
		<li>Perform morphological analysis evaluating the presence of cell suspension by light microscopy after 24 hr and 7 days of culture in flask.</li>
		<li>Analyze the samples of Dermis for positivity to the mesenchymal and hematopoietic cell markers, including CD146, CD34 and CD45 antigens by FACS analysis 6.</li>
		<li>Under laminar flow hood seed each micro-graft (obtained by the simultaneous disaggregation of eight, three or four uniform samples of skin tissue, Ded or Dermis respectively) and a correspondent small fragment of each tissue sample not totally disaggregated (&#62;50 micron in size after disaggregation process) on Columbia agar plate containing 5% sheep blood broth 100 &#181;l.</li>
		<li>Incubate the plate at 37 &#176;C for three days and perform microbiological analysis on Columbia agar plate in order to assess the sterility11.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Sen, C. K., 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=Human+skin+wounds:+a+major+and+snowballing+threat+to+public+health+and+the+economy.">Human skin wounds: a major and snowballing threat to public health and the economy.</a> Wound Repair Regen. 17, 763-771 (2009).</li><li>Ellis, E., Sinn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+homologous+bone+grafts+in+maxillofacial+surgery.">Use of homologous bone grafts in maxillofacial surgery.</a> J Oral Maxillofac Surg. 51, 1181-1193 (1993).</li><li>Nguyen, A., Pasyk, K. A., Bouvier, T. N., Hassett, C. A., Argenta, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+survival+of+autologous+adipose+tissue+taken+and+transplanted+by+different+techniques.">Comparative study of survival of autologous adipose tissue taken and transplanted by different techniques.</a> Plast Reconstr Sur. 85, 378-389 (1990).</li><li>Abuzeni, P. Z., Alexander, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+Autologous+Fat+Transplantation+With+Platelet+Rich+Plasma.">Enhancement of Autologous Fat Transplantation With Platelet Rich Plasma.</a> AJCS. 18 (2), 59-70 (2001).</li><li>Trovato, L., 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=A+New+Medical+Device+Rigeneracons+Allows+to+Obtain+Viable+Micro-Grafts+from+Mechanical+Disaggregation+of+Human+Tissues.">A New Medical Device Rigeneracons Allows to Obtain Viable Micro-Grafts from Mechanical Disaggregation of Human Tissues.</a> J Cell Physiol. , (2015).</li><li>Zanzottera, F., Lavezzari, E., Trovato, L., Icardi, A., Graziano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+Derived+Stem+Cells+and+Growth+Factors+Applied+on+Hair+Transplantation+Follow-Up+of+Clinical+Outcome.">Adipose Derived Stem Cells and Growth Factors Applied on Hair Transplantation Follow-Up of Clinical Outcome.</a> JCDSA. 4 (4), 268-274 (2014).</li><li>Giaccone, M., Brunetti, M., Camandona, M., Trovato, L., Graziano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Medical+Device,+Based+on+Rigenera+Protocol+in+the+Management+of+Complex+Wounds.">A New Medical Device, Based on Rigenera Protocol in the Management of Complex Wounds.</a> J Stem Cells Res, Rev &amp; Rep. 1 (3), 3 (2014).</li><li>Aimetti, M., Ferrarotti, F., Cricenti, L., Mariani, G. M., Romano, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+dental+pulp+stem+cells+in+periodontal+regeneration:+a+case+report.">Autologous dental pulp stem cells in periodontal regeneration: a case report.</a> Int J Periodontics Restorative Dent. 34 (3), s27-s33 (2014).</li><li>Graziano, A., Carinci, F., Scolaro, S., D'Aquino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodontal+tissue+generation+using+autologous+dental+ligament+micro-grafts:+case+report+with+6+months+follow-up.">Periodontal tissue generation using autologous dental ligament micro-grafts: case report with 6 months follow-up.</a> AOMS. 1 (2), 20 (2013).</li><li>Brunelli, G., Motroni, A., Graziano, A., D'Aquino, 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=Sinus+lift+tissue+engineering+using+autologous+pulp+micro-grafts:+A+case+report+of+bone+density+evaluation.">Sinus lift tissue engineering using autologous pulp micro-grafts: A case report of bone density evaluation.</a> J Indian Soc Periodontol. 17 (5), 644-647 (2013).</li><li>Ellner, P. D., Stoessel, C. J., Drakeford, E., Vasi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+culture+medium+for+medical+bacteriology.">A new culture medium for medical bacteriology.</a> Am J Clin Pathol. 45, 502-504 (1966).</li></ol>

The author Antonio Graziano is the Scientific Director of Human Brain Wave s.r.l. that produces and markets the Rigenera system. The author Letizia Trovato is a collaborator of Scientific Division of Human Brain Wave s.r.l.

This preliminary study showed that micro-grafts obtained by this protocol can be combined with collagen sponges, as already reported in other clinical applications, to optimize the efficacy of micro-grafts implants9, 10. In particular, this study reported the capacity of bio-complexes, constituted by micro-grafts and collagen sponges, to adjuvant the wound healing of a leg lesion after 30 days from clinical application. Furthermore, in vitro results provide evidence about the effectiveness of this protocol to disaggregate three different cutaneous tissues, maintaining an appreciable cell viability both immediately after the mechanical disaggregation and also after 24 hr and 7 days of culture.

The maintenance of cell viability after this kind of homogenization allows to obtain in only one step sterile autologous micro-grafts, avoiding extensive manipulation. This evidence is in agreement with other recent papers in which the efficacy of this protocol in vitro was tested also in other type of tissues, including periosteum, biopsy of cardiac atria and lateral rectus muscle of eyeball5. In particular, we observed in Ded and dermis samples an increased cell viability after 7 days of culture, probably due to the use of a medium supplemented with 10% fetal bovine serum.

In addition to the maintenance of cell viability, this system is safe and easy to use and in a few minutes is able to mechanically disaggregate different types of tissues preventing the disruption of cell structures, with respect to traditional methods of cell isolation, such as enzymatic digestion that require longer time of execution. Furthermore, this system is able to select cells with a cut-off of 50 microns and this aspect is very important in consideration of the success of injectable micro-grafts, because this range includes progenitor cells able to differentiate in many cells types and then ameliorate the lesion repair. This protocol allows us not only to obtain viable but also autologous micro-grafts, given that the donor subject is also the acceptor of these micro-grafts. The capacity of this system to obtain autologous micro-grafts was especially reported in the field of dentistry where it has been shown that transplantation of autologous Dental Pulp Stem Cells (DPSCs) in a non- contained infrabony defect contributed to periodontal repair and regeneration of atrophic maxilla5,6. The ability of these micro-grafts to&#160; improve the wound healing process was also previously reported in the management of complex wounds after surgical interventions or complications4.
Another advantage on the use of autologous and ready to use micro-grafts is certainly the keeping of sterile conditions in view of their subsequent implant on the skin lesions.

In conclusion, the protocol described in this paper showed the capacity to create human micro-graft tissue ready to use in clinical intervention to improve healing of acute/chronic skin wounds, such as those indicated in this study.&#160; In vitro results on the possibility to create viable micro-grafts have also been reported and this parameter is certainly significant to increase the percentage of success in the tissue repair. Taken together, the data showed that this new system could be considered a promising tool for clinicians who are in need of a rapid and safe instrument in the management of skin wounds.

At the moment, there are not particular modifications or limitations for the protocol in this specific application, given that the skin represents a tissue easy to use and in other studies we reported the efficacy of the same protocol in different skin lesions 6,7. We hope in the future to use this system on a large number of subjects to validate its clinical use in other clinical areas, (for example the orthopedics for bone regeneration or in vascular medicine for the ulcers treatment).

In the last years, several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery in order to provide one-step treatments for acute and chronic skin lesions. Moreover, the management of acute but mainly chronic wounds resulting from trauma, diabetes, infections, and other causes, remains challenging. There is mounting evidence that chronic wounds have become a serious global health issue, causing an enormous financial burden on healthcare systems worldwide1.

To increase the rate of success in the treatment of skin lesions, the absence of extensive manipulation (including cellular enhancement) and the maintenance of sterile conditions are essential, in order to create a cellular suspension that can be immediately applied on the damaged area of the patients, thereby avoiding a longer processing in cleanrooms such as Cell Factories. Starting from small skin biopsies, grinding, centrifugation and other separation methods (e.g., enzymatic or mechanical), are frequently used to obtain a cellular suspension, which can be cultured in a growth medium. All of these methods generally require a long time of execution, stressing the cell structures, and leading to a reduction of cell viability. Another significant aspect is to obtain an autologous cellular suspension ready to be used by clinicians, for example, to repair damaged areas. Furthermore, it is well established that autologous tissue grafts survive the transfer procedures to eventually survive in the recipient site by the principles of induction and conduction 2, 3. The ideal graft tissue should be readily available and have low antigenicity and donor site morbidity 4.

On the basis of these evidences, the first aim of this study was to create autologous bio-complexes suitable for clinical application in the tissue repair. For this purpose, we describe a new method to obtain autologous micro-grafts starting from cutaneous tissues which were disaggregated by this protocol. A case presentation is also herein described as a clinical application of autologous micro-grafts obtained by this protocol in combination with collagen sponges. This approach has already been reported to be efficient in the mechanical disaggregation of human tissues5 and it has been used clinically for grafts and regeneration of dermal tissues 6,7 as well as for regenerative therapies of connective tissues in oral-maxillofacial surgery 8-10.

In addition, the second aim of this study was the biological characterization of the cutaneous tissues after their disaggregation by this protocol. To this purpose, different homologous samples of cutaneous tissue derived from the trunk area of different multi-organ and/or multi-tissue donors were processed following National Rules on harvesting, processing and distributing tissues for transplantation (CNT 2013) at Emilia Romagna Regional Skin Bank.
CASE PRESENTATION:&#160; 
A 35-year-old female patient showing a complex trauma due to car accident was admitted to the Intensive Care Unit of Ancona Hospital. The patient showed an infection on the leg due to an open wound and a compound fracture stabilized with external fixation. Two radical debridement were performed and when the wound became clean after negative pressure therapy (V.A.C. therapy) and the periosteum appeared healthy, we applied the protocol after two months from recovery. After disaggregation with this system, the micro-grafts obtained were used to create bio-complexes with a collagen sponge which were subsequently implanted in order to investigate their efficacy on the lesion repair.

<table><tbody><tr><td>Rigenera Machine</td><td>Human Brain Wave</td><td>79210R</td><td></td></tr><tr><td>Rigeneracons</td><td>Human Brain Wave</td><td>79450S</td><td></td></tr><tr><td>MTT</td><td>Roche Diagnostic GmbH</td><td>11465007001</td><td></td></tr><tr><td>RPMI medium</td><td>PBI International</td><td>733-2292</td><td></td></tr><tr><td>DMEM medium</td><td>PBI International</td><td>F 0415</td><td></td></tr><tr><td>Antibiotics</td><td>Biological Industries PBI</td><td>03-038-1 &amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Antibodies for FACS Analysis</td><td>eBiosciences</td><td>code 12-1469 for CD146-P; code 17-0349&amp;nbsp; for CD34-APC&amp;nbsp;</td><td></td></tr><tr><td>Columbia agar</td><td>BioMerieux Company</td><td>43041</td><td></td></tr><tr><td>Condress&amp;reg;</td><td>Abiogen Pharma</td><td></td><td>collagen sponge used for bio-complexes</td></tr><tr><td>DMSO&amp;nbsp;</td><td>Bioniche Pharma USA LLC, Lake Forest, IL</td><td></td><td></td></tr><tr><td>NaCl solution</td><td>Fresenius Kabi, Bad Homburg, Germany</td><td></td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>Carlo Erba</td><td></td><td></td></tr></tbody></table>

tissue characterization after a new disaggregation method for skin micro-grafts generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

In this preliminary study, the first aim was to investigate the ability of human autologous micro-grafts combined with a biological support, such as collagen, to produce bio-complexes ready to use. These bio-complexes were implanted in a patient with a leg lesion caused by a car accident (Figure 2A) and a complete re-epithelialization associated with tissue repair after 30 days (Figure 2B) was observed. Moreover, the clinical follow-up showed a good texture and softness of the damaged area after 5 months (Figure 2C). In parallel to clinical application, in vitro studies were also performed to evaluate the cell viability of different cutaneous tissues such as skin tissue, Ded and Dermis before and after disaggregation by this system. In particular, taking into account the different thickness of each type of tissue, the threshold of eight, four and three biopsies that can be processed in a single step for skin tissue, Ded and Dermis, respectively, was established. The established number of biopsies were then disaggregated at four different times as indicated in the Table 1, according to their biological characteristics in order to identify the optimal condition of disaggregation to maintain a good cell viability.
Although the tissue processing itself inevitably induced an impairment of cell viability compared to intact tissue, the mechanical disaggregation performed with this system seems to maintain a mean value of cell viability of 30% in all samples of skin, Ded and dermis evaluated at different times (Figure 3A), with respect to intact tissue (Figure 3B) (mean of 92 OD/gr for intact skin tissue and 29 OD/gr after disaggregation; from 22 OD/gr to 7 OD/gr for intact Ded with respect to disaggregation and finally from 16 OD/gr to 2.7 OD/gr for intact dermis with respect to disaggregation). Thus, these preliminary results show that this system is able to maintain appreciable levels of cell viability after immediate disaggregation. The effect of disaggregation on cell viability was also investigated on tissue samples cultured for 24 hr or 7 days and variable results were observed. In particular, no substantial variation of cell viability in skin tissue samples was observed independently by time of homogenization and culture compared to starting time (T0) (Figure 4A). On the other hand, a reduced viability of Ded samples after 24 hr of culture compared to starting time (T0) was observed but surprisingly the cell viability was restored after 7 days of culture (Figure 4B). Similar results were also observed for samples of Dermis (Figure 4C). Each sample was evaluated in duplicate and values reported in the Table 2.
On the basis of these results, we identified the suitable time of disaggregation to maintain cell viability for three types of cutaneous tissue as reported in Table 3. In addition to cell viability, the morphological aspect of cultured cellular suspension was also evaluated, identifying single cells after 24 hr from tissue disaggregation, while after 7 days there residues of fibers in both skin tissue and Ded/Dermis samples were observed (Figure 5 A-B). In addition, a cell characterization by flow cytometry analysis was performed in a sample of dermis after its homogenization: a heterogeneous pool of cells composed by several cellular types, including endothelial cells and mesenchymal stem cells was identified (Figure 6). Furthermore, the absence of bacterial growth was identified in all disaggregated samples opportunely seeded on Agar dishes, evidencing the sterility of experimental procedure (Figure 7).
<img alt="Figure 1" src="/files/ftp_upload/53579/53579fig1.jpg" /> 
Figure 1. Bio-complexes Building Tissue Disrupting System (A) and Collection of a Small Piece of Derma from the Lesion Area of the Patient that was Subsequently Disaggregated with Tissue Disruptors. The bio-complexes were obtained combining the micro-grafts on collagen sponges to obtain human patches ready to use for lesion repair (C). (D) Hematoxylin &#38; eosin (H&#38;E) staining of a representative bio-complex cultured for three days in DMEM medium and observed to microscopy <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53579/53579fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53579/53579fig2.jpg" /> 
Figure 2. Bio-complexes Application on Leg Lesion The bio-complexes composed by collagen and micro-grafts obtained from the patient were applied on the leg lesion (A) and the wound was evaluated after 30 days (B) and 5 months (C). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53579/53579fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53579/53579fig3.jpg" /> 
Figure 3. Cell Viability Immediately after Disaggregation at Different Times of Three Type of Cutaneous Tissues (A) with respect to Intact Cutaneous Tissues (B). The Skin, Ded and Dermis tissues derived from four different donors were disaggregated with tissue disruptors as indicate in the appropriate section in the text. Cell viability was assessed by MTT test performed in duplicate for each tissue sample. The graph is representative of four different experiments performed in duplicate for each sample. The results are expressed as a ratio between Optical Density (OD) and grams (gr) of tissue; the standard deviation was calculated on the ratio between Optical Density (OD) and grams (gr) of tissue. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53579/53579fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53579/53579fig4.jpg" /> 
Figure 4. Cell Viability Levels of a Disaggregated Sample of Skin Tissue (A), Ded (B) and Dermis (C) to Starting Time (T0) and after Subculture for 24 hr and 7 Days. The tissues were homogenized with tissue disruptors as indicate in the appropriate section in the text. The disaggregated samples were subsequently cultured for 24 hr and 7 days after the disaggregation and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics at 37 &#176;C in an atmosphere of 5% CO2/air. Cell viability was assessed by MTT as previously described. The graph is representative of an experiment performed in duplicate for each sample of skin tissue, Ded and Dermis derived from a single donor. The results are expressed as ratio between Optical Density (OD) and grams (gr) of tissue. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53579/53579fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53579/53579fig5.jpg" /> 
Figure 5. Morphological Analysis of Disaggregated Skin Tissue (A) and Ded/Dermis (B) after 24 hr and 7 Days of Culture. Morphological analysis was performed using light microscopy after 24 hr and 7 days of culture in the presence of RPMI medium on skin tissue and Ded/dermis tissues. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53579/53579fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53579/53579fig6.jpg" /> 
Figure 6. Cell Characterization by Flow Cytometry Analysis. After mechanical disaggregation, micro-grafts obtained by dermis were put in culture and after 7 days were analyzed for positivity to mesenchymal and hematopoietic cell marker, including CD146, CD34 and CD45 antigens. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53579/53579fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/53579/53579fig7.jpg" /> 
Figure 7. Microbiological Analysis of Disaggregated Skin Tissue, Ded and Dermis Samples. Small fragments of disaggregated tissue samples were seeded on Columbia agar added of 5% sheep blood (BioMerieux Company) under laminar flow hood and incubated at 37 &#176;C for three days to perform the microbiological analysis and assess the sterility of procedure. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53579/53579fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>TISSUE SAMPLES</td>
			<td>DISAGGREGATION TIMES (sec/min)</td>
		</tr>
		<tr>
			<td>Skin</td>
			<td>30 sec</td>
		</tr>
		<tr>
			<td></td>
			<td>1 min</td>
		</tr>
		<tr>
			<td></td>
			<td>2 min</td>
		</tr>
		<tr>
			<td></td>
			<td>5 min</td>
		</tr>
		<tr>
			<td>Ded</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td></td>
			<td>2 min</td>
		</tr>
		<tr>
			<td></td>
			<td>5 min</td>
		</tr>
		<tr>
			<td></td>
			<td>10 min</td>
		</tr>
		<tr>
			<td>Dermis</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td></td>
			<td>2 min</td>
		</tr>
		<tr>
			<td></td>
			<td>5 min</td>
		</tr>
		<tr>
			<td></td>
			<td>10 min</td>
		</tr>
	</tbody>
</table>
Table 1. Schematic Representation of the Four Different Times used for Disaggregation of Skin, Ded and Dermis. Eight, four and three punch biopsies of Skin, Ded and Dermis respectively were disaggregated at four different times, according to their biological characteristics.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td colspan="6">Skin tissue</td>
		</tr>
		<tr>
			<td colspan="2">To</td>
			<td colspan="2">24 hr</td>
			<td colspan="2">7 days</td>
		</tr>
		<tr>
			<td>2&#8217;</td>
			<td>5&#8217;</td>
			<td>2&#8217;</td>
			<td>5&#8217;</td>
			<td>2&#8217;</td>
			<td>5&#8217;</td>
		</tr>
		<tr>
			<td>37.1</td>
			<td>41.07143</td>
			<td>41.07143</td>
			<td>65.78571</td>
			<td>41.14286</td>
			<td>37.42857</td>
		</tr>
		<tr>
			<td>35.8</td>
			<td>43.82143</td>
			<td>36.60714</td>
			<td>66.5</td>
			<td>41.46429</td>
			<td>38.67857</td>
		</tr>
		<tr>
			<td colspan="6">Ded</td>
		</tr>
		<tr>
			<td colspan="2">T0</td>
			<td colspan="2">24 hr</td>
			<td colspan="2">7 days</td>
		</tr>
		<tr>
			<td>5&#39;</td>
			<td>10&#39;</td>
			<td>5&#39;</td>
			<td>10&#39;</td>
			<td>5&#39;</td>
			<td>10&#39;</td>
		</tr>
		<tr>
			<td>6,649485</td>
			<td>8.237113</td>
			<td>5.463918</td>
			<td>5.731959</td>
			<td>7.835052</td>
			<td>7.85567</td>
		</tr>
		<tr>
			<td>6,845361</td>
			<td>8,360825</td>
			<td>5.257732</td>
			<td>5.989691</td>
			<td>8</td>
			<td>7.938144</td>
		</tr>
		<tr>
			<td colspan="6">Dermis</td>
		</tr>
		<tr>
			<td colspan="2">To</td>
			<td colspan="2">24 hr</td>
			<td colspan="2">7 days</td>
		</tr>
		<tr>
			<td>10&#39;</td>
			<td>5&#39;</td>
			<td>10&#39;</td>
			<td>5&#39;</td>
			<td>10&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>1.690909</td>
			<td>2.77193</td>
			<td>0.293898</td>
			<td>0.786704</td>
			<td>2.880952</td>
			<td>2.869048</td>
		</tr>
		<tr>
			<td>1.736364</td>
			<td>2.830409</td>
			<td>0.306351</td>
			<td>0.814404</td>
			<td>2.940476</td>
			<td>2.928571</td>
		</tr>
	</tbody>
</table>
Table 2. Range of Values Expressed in OD/gr for One Biological Replicate. Values of cell viability from skin tissue, Ded and dermis evaluated to starting time and after mechanical disaggregation to indicated times. The results are representative of one experiment performed in duplicate and expressed as ratio between Optical Density (OD) and grams (gr) of tissue.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Specimen</td>
			<td>Disaggregation Time </td>
		</tr>
		<tr>
			<td>Skin tissue</td>
			<td>5 min</td>
		</tr>
		<tr>
			<td>Ded</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>Derma</td>
			<td>2 min</td>
		</tr>
	</tbody>
</table>
Table 3. Schematic Representation of the Best Time of Disaggregation to Maintain a Good Cell Viability in Different Tissue Samples. Skin, Ded and Dermis tissue samples show the optimal cell viability after 5, 1 and 2 min of disaggregation, respectively.

Watch this Scientific Journal Video about Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation at JoVE.com

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

tissue-characterization-after-new-disaggregation-method-for-skin

Universidad San Sebastian

Research

JoVE Journal

Medicine

21.5K Views.  Burns Centre and Emilia Romagna Regional Skin Bank. The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

Video: Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Developmental Biology

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

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Bioengineering

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Immunology and Infection

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.

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 ...

Decellularization for the Preparation of Highly Preserved Human Acellular Skin Matrix for Regenerative Medicine

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of surrounding cells due to its content of diverse bioactive molecules. Due to these characteristics, the ECM has been recently considered a promising candidate for the creation of biological scaffolds to boost tissue regeneration. Emerging studies have demonstrated that decellularized human tissues could resemble the native ECM in their structural and biochemical profiles, preserving the three-dimensional (3D) architecture and the content of fundamental biological molecules. Hence, decellularized ECM can be employed to promote tissue remodeling, repair, and functional reconstruction of many organs. Selecting the appropriate decellularization procedure is crucial to obtain acellular tissues that retain the characteristics of the ideal microenvironment for cells. 
The protocol described here provides a detailed step-by-step description of the decellularization method to obtain a reproducible and effective cell-free biological ECM. Skin fragments from patients undergoing plastic surgery were scaled down and decellularized using a combination of sodium dodecylsulfate (SDS), Triton X-100, and antibiotics. To promote the regular and homogeneous transport of the solution through the samples, they were enclosed in embedding cassettes to ensure protection from mechanical insults. After the decellularization procedure, the snow-white color of skin fragments indicated complete and successful decellularization. Additionally, decellularized samples showed an intact and well-preserved architecture. The results suggest that the proposed decellularization method was effective, fast, and reproducible and protected samples from architectural damages.

Decellularized human skin is suitable for tissue regeneration. A major issue of decellularization is the preservation of the native architecture, along with the appropriate content of structural proteins, glycosaminoglycans (GAGs), and growth factors. The method proposed allows fast and effective decellularization, producing decellularized skin with well-preserved native features.

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Biology

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report.

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Summary

Explore More Videos

Chapters in this video

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

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

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Apparatus for Harvesting Tissue Microcolumns

Decellularization for the Preparation of Highly Preserved Human Acellular Skin Matrix for Regenerative Medicine

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo