Name: production of autologous platelet-rich plasma for boosting in vitro human fibroblast expansion
Uploaded: 2021-02-24T12:30:00
Description: Watch this Scientific Journal Video about Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion at JoVE.com

We thank Mr Gr&#233;gory Schneiter for technical assistance with flow cytometry data; Professor Muriel Cuendet (Laboratory of Pharmacognosy, School of Pharmaceutical Sciences, and University of Geneva) for allowing the use of the Attune flow cytometer and the Cytation 3 high-throughput microscope; Professor Brigitte Pittet for scientific advices.

The study protocol complied with the Declaration of Helsinki, and all patients provided written informed consent before participating in the study. Skin samples are obtained from healthy women undergoing abdominoplasty in the Plastic, Reconstructive and Aesthetic Surgery Department at Geneva University Hospitals (Geneva, Switzerland). The procedure conforms to the principles of the Declaration of Helsinki and was approved by the local institutional ethics committee (protocol #3126).
1. Preparati...

<ol>
	<li>Kumar, S., Mahajan, B. B., Kaur, S., Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+therapies+in+dermatology.">Autologous therapies in dermatology.</a> The Journal of Clinical and Aesthetic Dermatology. 7 (12), 38-45 (2014).</li><li>Martin, I., 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=The+survey+on+cellular+and+engineered+tissue+therapies+in+Europe+in+2009.">The survey on cellular and engineered tissue therapies in Europe in 2009.</a> Tissue Engineering. Part A. 17 (17-18), 2221-2230 (2011).</li><li>Stunova, A., Vistejnova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dermal+fibroblasts-A+heterogeneous+population+with+regulatory+function+in+wound+healing.">Dermal fibroblasts-A heterogeneous population with regulatory function in wound healing.</a> Cytokine &amp; Growth Factor Reviews. 39, 137-150 (2018).</li><li>Thangapazham, R. L., Darling, T. N., Meyerle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+skin+properties+with+autologous+dermal+fibroblasts.">Alteration of skin properties with autologous dermal fibroblasts.</a> International Journal of Biological Sciences. 15 (5), 8407-8427 (2014).</li><li>Costa-Almeida, R., Soares, R., Granja, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+as+maestros+orchestrating+tissue+regeneration.">Fibroblasts as maestros orchestrating tissue regeneration.</a> Journal of Tissue Engineering and Regenerative Medicine. 12 (1), 240-251 (2018).</li><li>Weiss, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+cell+therapy:+will+it+replace+dermal+fillers.">Autologous cell therapy: will it replace dermal fillers.</a> Facial Plastic Surgery Clinics of North America. 21 (2), 299-304 (2013).</li><li>Ichim, T. E., O'Heeron, P., Kesari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+as+a+practical+alternative+to+mesenchymal+stem+cells.">Fibroblasts as a practical alternative to mesenchymal stem cells.</a> Journal of Translational Medicine. 16 (1), 212 (2018).</li><li>Gstraunthaler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternatives+to+the+use+of+fetal+bovine+serum:+serum-free+cell+culture.">Alternatives to the use of fetal bovine serum: serum-free cell culture.</a> ALTEX. 20 (4), 275-281 (2003).</li><li>Karnieli, O., 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+consensus+introduction+to+serum+replacements+and+serum-free+media+for+cellular+therapies.">A consensus introduction to serum replacements and serum-free media for cellular therapies.</a> Cytotherapy. 19 (2), 155-169 (2017).</li><li>van der Valk, 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=Fetal+Bovine+Serum+(FBS):+Past+-+Present+-+Future.">Fetal Bovine Serum (FBS): Past - Present - Future.</a> ALTEX. 35 (1), 99-118 (2018).</li><li>Cavallo, 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=Platelet-Rich+Plasma:+The+Choice+of+Activation+Method+Affects+the+Release+of+Bioactive+Molecules.">Platelet-Rich Plasma: The Choice of Activation Method Affects the Release of Bioactive Molecules.</a> BioMed Research International. 2016, 6591717 (2016).</li><li>Peng, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-Rich+Plasma+for+Skin+Rejuvenation:+Facts,+Fiction,+and+Pearls+for+Practice.">Platelet-Rich Plasma for Skin Rejuvenation: Facts, Fiction, and Pearls for Practice.</a> Facial Plastic Surgery Clinics of North America. 27 (3), 405-411 (2019).</li><li>Fadadu, P. P., Mazzola, A. J., Hunter, C. W., Davis, T. T. <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+concentration+yields+in+commercially+available+platelet-rich+plasma+(PRP)+systems:+a+call+for+PRP+standardization.">Review of concentration yields in commercially available platelet-rich plasma (PRP) systems: a call for PRP standardization.</a> Regional Anesthesia and Pain Medicine. 44, 652-659 (2019).</li><li>Berndt, S., Turzi, A., Pittet-Cuenod, B., Modarressi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+Platelet-Rich+Plasma+(CuteCell+PRP)+Safely+Boosts+In+Vitro+Human+Fibroblast+Expansion.">Autologous Platelet-Rich Plasma (CuteCell PRP) Safely Boosts In Vitro Human Fibroblast Expansion.</a> Tissue Engineering. Part A. 25 (21-22), 1550-1563 (2019).</li><li>Zeng, 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=Preclinical+safety+studies+on+autologous+cultured+human+skin+fibroblast+transplantation.">Preclinical safety studies on autologous cultured human skin fibroblast transplantation.</a> Cell Transplantation. 23 (1), 39-49 (2014).</li><li>Lee, E. C. 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=Efficacy+of+Autologous+Cultured+Fibroblast+Cells+as+a+Treatment+for+Patients+with+Facial+Contour+Defects:+A+Clinical+Replication+Study.">Efficacy of Autologous Cultured Fibroblast Cells as a Treatment for Patients with Facial Contour Defects: A Clinical Replication Study.</a> Journal of Cosmetics, Dermatological Sciences and Applications. 7, 306-317 (2017).</li><li>Eca, L. P., Pinto, D. G., de Pinho, A. M., Mazzetti, M. P., Odo, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+fibroblast+culture+in+the+repair+of+aging+skin.">Autologous fibroblast culture in the repair of aging skin.</a> Dermatologic Surgery. 38 (2), 180-184 (2012).</li><li>Nilforoushzadeh, M. 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=Autologous+fibroblast+suspension+for+the+treatment+of+refractory+diabetic+foot+ulcer.">Autologous fibroblast suspension for the treatment of refractory diabetic foot ulcer.</a> Indian Journal of Dermatology, Venereology and Leprology. 82 (1), 105-106 (2016).</li><li>Cowper, 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=Human+Platelet+Lysate+as+a+Functional+Substitute+for+Fetal+Bovine+Serum+in+the+Culture+of+Human+Adipose+Derived+Stromal/Stem+Cells.">Human Platelet Lysate as a Functional Substitute for Fetal Bovine Serum in the Culture of Human Adipose Derived Stromal/Stem Cells.</a> Cells. 8 (7), 724 (2019).</li><li>Atashi, F., Jaconi, M. E., Pittet-Cuenod, B., Modarressi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+platelet-rich+plasma:+a+biological+supplement+to+enhance+adipose-derived+mesenchymal+stem+cell+expansion.">Autologous platelet-rich plasma: a biological supplement to enhance adipose-derived mesenchymal stem cell expansion.</a> Tissue Engineering Part C: Methods. 21 (3), 253-262 (2015).</li><li>Martinez-Zapata, M. 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=Autologous+platelet-rich+plasma+for+treating+chronic+wounds.">Autologous platelet-rich plasma for treating chronic wounds.</a> The Cochrane Database of Systematic Reviews. (5), (2016).</li><li>Cervelli, V., 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=Use+of+platelet-rich+plasma+and+hyaluronic+acid+in+the+loss+of+substance+with+bone+exposure.">Use of platelet-rich plasma and hyaluronic acid in the loss of substance with bone exposure.</a> Advances in Skin &amp; Wound Care. 24 (4), 176-181 (2011).</li><li>Nicoli, F., 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=Severe+hidradenitis+suppurativa+treatment+using+platelet-rich+plasma+gel+and+Hyalomatrix.">Severe hidradenitis suppurativa treatment using platelet-rich plasma gel and Hyalomatrix.</a> International Wound Journal. 12 (3), 338-343 (2015).</li><li>Gentile, P., Bottini, D. J., Spallone, D., Curcio, B. C., Cervelli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+platelet-rich+plasma+in+maxillofacial+surgery:+clinical+evaluation.">Application of platelet-rich plasma in maxillofacial surgery: clinical evaluation.</a> The Journal of Craniofacial Surgery. 21 (3), 900-904 (2010).</li><li>Saleem, 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=Adjunctive+Platelet-Rich+Plasma+(PRP)+in+Infrabony+Regenerative+Treatment:+A+Systematic+Review+and+RCT's+Meta-Analysis.">Adjunctive Platelet-Rich Plasma (PRP) in Infrabony Regenerative Treatment: A Systematic Review and RCT's Meta-Analysis.</a> Stem Cells International. 2018, 9594235 (2018).</li><li>Everts, P. A., Pinto, P. C., Girao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+pure+platelet-rich+plasma+injections+for+facial+skin+rejuvenation:+Biometric+instrumental+evaluations+and+patient-reported+outcomes+to+support+antiaging+effects.">Autologous pure platelet-rich plasma injections for facial skin rejuvenation: Biometric instrumental evaluations and patient-reported outcomes to support antiaging effects.</a> Journal of Cosmetic Dermatology. 18 (4), 985-995 (2019).</li><li>Fiaschetti, V., 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=Magnetic+resonance+imaging+and+ultrasound+evaluation+after+breast+autologous+fat+grafting+combined+with+platelet-rich+plasma.">Magnetic resonance imaging and ultrasound evaluation after breast autologous fat grafting combined with platelet-rich plasma.</a> Plastic and Reconstructive Surgery. 132 (4), 498-509 (2013).</li><li>Gentile, P., Scioli, M. G., Orlandi, A., Cervelli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+Reconstruction+with+Enhanced+Stromal+Vascular+Fraction+Fat+Grafting:+What+Is+the+Best+Method.">Breast Reconstruction with Enhanced Stromal Vascular Fraction Fat Grafting: What Is the Best Method.</a> Plastic and Reconstructive Surgery. 3 (6), 406 (2015).</li><li>Modarressi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platlet+Rich+Plasma+(PRP)+Improves+Fat+Grafting+Outcomes.">Platlet Rich Plasma (PRP) Improves Fat Grafting Outcomes.</a> World Journal of Plastic Surgery. 2 (1), 6-13 (2013).</li><li>Muraglia, 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=Culture+Medium+Supplements+Derived+from+Human+Platelet+and+Plasma:+Cell+Commitment+and+Proliferation+Support.">Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support.</a> Frontiers in Bioengineering and Biotechnology. 5, 66 (2017).</li><li>Ferrao, A. V., Mason, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+heparin+on+cell+proliferation+and+type-I+collagen+synthesis+by+adult+human+dermal+fibroblasts.">The effect of heparin on cell proliferation and type-I collagen synthesis by adult human dermal fibroblasts.</a> Biochimica et Biophysica Acta. 1180 (3), 225-230 (1993).</li><li>Gonzalez-Delgado, P., Fernandez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypersensitivity+reactions+to+heparins.">Hypersensitivity reactions to heparins.</a> Current Opinion in Allergy and Clinical Immunology. 16 (4), 315-322 (2016).</li><li>Atashi, F. S. B., Nayernia, Z., Pittet-Cu&#233;nod, B., Modarressi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+Rich+Plasma+Promotes+Proliferation+of+Adipose+Derived+Mesenchymal+Stem+Cells+via+Activation+of+AKT+and+Smad2+Signaling+Pathways.">Platelet Rich Plasma Promotes Proliferation of Adipose Derived Mesenchymal Stem Cells via Activation of AKT and Smad2 Signaling Pathways.</a> Stem Cell Research &amp; Therapy. 5 (8), (2015).</li></ol>

The advantages of using autologous fibroblasts as a natural alternative compared to other filler materials in wound cell therapy include good biocompatibility, minimal side effects, and easiness of harvesting and use. However, before using these therapeutics in a daily clinical setting, proper preclinical studies are necessary to identify the growth features and assess the biological function and safety of isolated fibroblasts both before and after transplantation. Thus, directly after the isolation process, in vitro exp...

Regenerative medicine aims to heal or replace tissues and organs damaged by age, disease, or trauma as well as correct congenital defects. In autologous therapy, cells or tissue are withdrawn from a patient, expanded or modified, then reintroduced to the donor. This form of therapeutics has broad potential in the field of dermatology1. In autologous fibroblast therapy, a patient&#8217;s fibroblasts are cultured and reinjected to treat wrinkles, rhytids, or acne scars. As fibroblasts are the main functional cells in the dermis, injection of autologous fibroblasts may be more beneficial than other therapies in facial rejuvenation2.
In the skin, fibroblasts are responsible for the synthesis and secretion of extracellular proteins (i.e., collagen, elastin, hyaluronic acid, and glycosaminoglycans). They also release growth factors that regulate cell function, migration, and cell-matrix/cell-cell interactions in normal skin homeostasis and wound healing3. Dermal fibroblasts have already been introduced as a potential clinical cell therapy for skin wound healing4, tissue regeneration5, or dermal fillers in esthetic and plastic surgery procedures6. Some studies even suggest that, in the context of regenerative medicine, fibroblasts may be a more practical and effective cell therapy than mesenchymal stem cells7.
To obtain a sufficient number of fibroblasts for clinical applications, cell expansion is usually mandatory. Ex vivo/in vitro cell culture requires basal medium supplemented with growth factors, proteins, and enzymes to support cell adhesion and proliferation. Fetal bovine serum (FBS) is a common supplement for cell culture media, because fetal blood 1) is rich in growth factors compared to adult blood and 2) presents a low antibody content8. As cell therapy progresses, there are concerns about the safety of classical cell culture conditions in which FBS is added to the culture medium. Furthermore, there is now a tendency to replace FBS with alternatives9. Several FBS substitutes have shown promising results10.
The platelet-rich plasma (PRP) alternative has been selected here, and we have developed a medical device to produce a standardized preparation of PRP, named CuteCell-PRP. The intended use of this device is the preparation of autologous PRP to be used as a culture media supplement for in vitro expansion of autologous cells under GMP conditions.
PRP is defined as a concentrated platelet suspension in plasma. Because there are numerous preparation protocols, which differ in 1) the amount of blood needed, 2) types of devices used, and 3) centrifugation protocol, the resulting platelet concentrations vary from slightly above to more than 10x the blood baseline value. In addition, PRP preparations contain variable levels of red and white blood cell contamination. The terminology &#8220;PRP&#8221; is thus used to describe products that vary greatly in their biological composition and potential therapeutic effects.
In most studies, FBS substitution is achieved using different concentrations of PRP that is activated (by thrombin or calcium). This artificial activation provokes an immediate and important release of platelet growth factors from 15 min up to 24 h11. Therefore, it is believed that platelet activation is undesirable for applications in cell cultures, in which the slow release of growth factors from gradual platelet degranulation is required.
PRP therapy involves the preparation of autologous platelets in concentrated plasma12. The optimal platelet concentration is unclear, and a broad range of commercial devices are available to prepare PRP13. This lack of standardization results from inconsistency among studies and has led to a black box regarding the dosage and timing of injection. This protocol describes a procedure to obtain autologous PRP using this dedicated PRP device to expand skin fibroblasts in a 100% autologous ex vivo culture model.

<table>
	<tbody>
		<tr>
			<td>96 well black clear flat bottom</td>
			<td>BD Falcon</td>
			<td>353219</td>
			<td>32/case</td>
		</tr>
		<tr>
			<td>Cell trace Violet Dye</td>
			<td>Thermo Fischer Scientific</td>
			<td>C34557</td>
			<td>180 assays</td>
		</tr>
		<tr>
			<td>CuteCell PRP</td>
			<td>Regen Lab SA</td>
			<td>CC-PRP-3T</td>
			<td>3 tubes per package</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>1 mg</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>52400-025</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Glutamine 200 mM</td>
			<td>Gibco</td>
			<td>25030024</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Hematology Counter</td>
			<td>Sysmex</td>
			<td>KK-21N</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin 5000E Liquemine</td>
			<td>Drossapharm AG</td>
			<td></td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>HEPES Buffer Solution 1M</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Liberase DH</td>
			<td>Roche</td>
			<td>5401054001</td>
			<td>2x 5 mg per package</td>
		</tr>
		<tr>
			<td>MEM NEAA 100x</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Na Pyruvate 1mg/mL</td>
			<td>Gibco</td>
			<td>11360-039</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Penicillin streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Phalloidin alexa Fluor 488</td>
			<td>Molecular Probes</td>
			<td>A12379</td>
			<td>300 units</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Trypsin 1x 0.25%</td>
			<td>Gibco</td>
			<td>25050-014</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Trypsin EDTA 0.25%</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td>100 mL</td>
		</tr>
	</tbody>
</table>

production of autologous platelet-rich plasma for boosting in vitro human fibroblast expansion

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient&#8217;s own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.

This patented technology is a simple, fast, and reproducible medical device used to produce standardized PRP preparations. It is a one-step, fully closed system that allows the preparation of PRP from venous whole blood after 5 min of centrifugation at 1,500 x g (due to the separating gel technology). The PRP obtained after centrifugation is cleared from red and white blood cells, which sit below the gel. After several tube inversions, the platelets that are on top of the gel are resuspended in the plasma, and t...

Watch this Scientific Journal Video about Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion at JoVE.com

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is ...

This protocol is the autologous system for human fibroblast culture with the patient's own platelet-rich plasma and a standardized preparation for five minutes. PRP promotes a massive cell expansion of fibroblast in culture. I would recommend you to use this method using PRP as a cell supplement when you need massive creek and safe cell expansion for autologous cell therapies.When you attempt this protocol, I would recommend you to check the PRP integrity. Your plasma has to be very clean with absence of red blood cells. When you transfer it, you have to do it very carefully.And concerning the digestion of your tissue, it has to be very clean before proceeding. To begin, carefully turn the blood-filled platelet-rich plasma, or PRP, tubes upside down three times to mix the blood with anticoagulant. Centrifuge the blood samples at 1500g for five minutes in a fixed 45-degree-angled rotor and balance the tubes.After centrifugation, the fractionated blood has red and white blood cells trapped under the separating gel and platelets on the surface of the gel. Gently invert the PRP tube 20 times to resuspend the platelet deposit in the plasma supernatant. Ensure that the platelets are fully detached from the gel and the plasma should change from clear and transparent to turbid.Collect the plasma solution from the tube using a transfer device connected to a 10-milliliter syringe. Place the skin samples in a 50-milliliter polypropylene tube with PBS and shake the tube gently. If the skin sample is relatively large, place it on the lid of a 150-square-centimeter tissue culture dish with the epidermal side down.Remove the subcutaneous fat tissue using a pair of forceps and scissors. To prevent the tissue from drying out, rinse the tissue every few minutes in PBS. When fat removal is complete, use a scalpel to cut the skin samples into strips, approximately 0.5 by 1.5 centimeters.Next, add five milliliters of Collagenase/Dispase mix to a sterile 15-milliliter tube. Transfer the cut tissue to the tube. Ensure that the tissue pieces are submerged in the solution and cap the tube securely.Place the tubes in an incubator at 37 degrees Celsius with orbital shaking for 150 minutes. After incubation, move the tube containing the tissue to the biosafety cabinet. Place the lid of a sterile 100-millimeter culture dish upside down in the biosafety cabinet.Transfer the tissue suspension to the bottom of the 100-millimeter culture dish without splashing. If any pieces of tissue remain in the tube, use a sterile one-milliliter pipette or sterile forceps to transfer the pieces to the bottom of the culture dish. With the epidermis facing up, hold the dermis with a pair of forceps.Grasp the edge of the epidermis with another pair of forceps and peel the dermis and epidermis apart. Keeping the separated pieces on the same lid, place the dermal pieces in a new 100-millimeter culture dish containing PBS. Next, using laboratory forceps, transfer the dermal pieces to a 15-milliliter polypropylene tube containing three milliliters of 0.3%Trypsin PBS.Incubate the tube in a 37-degree-Celsius water bath and mix the contents every two to three minutes by inverting the tube several times. After 10 to 20 minutes, stop the reaction by adding three to five milliliters of ice-cold complete growth medium. Vortex the tube vigorously several times.Filter the suspension, which contains the fibroblasts, through an 85-micron nylon mesh into a 50-milliliter tube. Centrifuge the fibroblast solution at 150g for 10 minutes. Aspirate the supernatant and resuspend the cell pellet in 100 to 200 microliters of complete growth medium for cell count.Plate the cells in a 25-square-centimeter tissue culture flask. Then, incubate the flask at 37 degrees Celsius. Change the medium which contains non-adherent cells on the following day, and again, every three to four days.When the fibroblasts reach 70 to 80%confluency, wash them with PBS and add Trypsin-EDTA solution. After incubating the flask for three minutes at 37 degrees Celsius, stop the reaction by adding three to five milliliters of warm complete growth medium. Then, centrifuge the cell suspension at 200g for five minutes.Remove the supernatant by aspiration. Culture the fibroblasts in PRP medium and incubate at 37 degrees Celsius. The minimum cell density recommended is 4, 000 viable cells per centimeter squared.A dedicated medical device was used to produce PRP by processing whole blood from 10 different patients. The device removed the majority of the red blood cells and white blood cells. The mean platelet concentration in the PRP was approximately 50%higher than the platelet concentration in whole blood.Autologous fibroblasts were cultivated in medium with 10%FBS or in media with PRP concentrations ranging from one to 50%After seven days of culture, without changing the medium, the cultures primed with 20%PRP exhibited a higher number of viable fibroblasts. PRP had potent effects as a proliferation booster up to 7.7 fold compared to FBS. Phalloidin staining showed that the morphological change observed upon PRP activation was related to F-actin reorganization, which is a signature of fibroblast activation.A major concern of rapid cell expansion is the risk of genomic modification. In order to assess this, you can perform comparative genomic hybridization assay to assess the genomic stability of your cells. The main advantage of this technique is to replace FBS by a PRP, which is a more potent autologous biological supplement.With this, researcher can grow more cell faster without any addition of a xenogeneic substances. We may use this technique for other cell types that need ex vivo expansion before clinical application, for example, keratinocyte or mesenchymal stem cell.

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Research

JoVE Journal

Bioengineering

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Area-based Image Analysis Algorithm for Quantification of Macrophage-fibroblast Cocultures

Quantification of cells is necessary for a wide range of biological and biochemical studies. Conventional image analysis of cells typically employs either fluorescence detection approaches, such as immunofluorescent staining or transfection with fluorescent proteins or edge detection techniques, which are often error-prone due to noise and other non-idealities in the image background. 
We designed a new algorithm that could accurately count and distinguish macrophages and fibroblasts, cells of different phenotypes that often colocalize during tissue regeneration. MATLAB was used to implement the algorithm, which differentiated distinct cell types based on differences in height from the background. A primary algorithm was developed using an area-based method to account for variations in cell size/structure and high-density seeding conditions. 
Non-idealities in cell structures were accounted for with a secondary, iterative algorithm utilizing internal parameters such as cell coverage computed using experimental data for a given cell type. Finally, an analysis of coculture environments was carried out using an isolation algorithm in which various cell types were selectively excluded based on the evaluation of relative height differences within the image. This approach was found to accurately count cells within a 5% error margin for monocultured cells and within a 10% error margin for cocultured cells.

We present a method, which utilizes a generalizable area-based image analysis approach to identify cell counts. Analysis of different cell populations exploited the significant cell height and structure differences between distinct cell types within an adaptive algorithm.

Quantification of cells is necessary for a wide range of biological and biochemical studies. Conventional image analysis of cells typically employs either ...