Name: Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion (Video) | JoVE Journal
Uploaded: 2021-02-24T12:30:00.000+00:00
Duration: 8 min 34 s
Description: Learn to produce autologous platelet-rich plasma for boosting human fibroblast expansion in vitro. This standardized five-minute protocol enables safe, massive cell growth for regenerative medicine applications.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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. Preparation of PRP
NOTE: The CuteCell-PRP tubes (Table of Materials) are designed for the rapid preparation of PRP from a small volume of the patient&#8217;s blood in a closed circuit system.
<ol>
	<li>Collection of whole blood 
	NOTE: Harvest autologous blood from a peripheral vein of the arm using a butterfly needle directly connected to the PRP tube, according to the collection protocol of the medical institute. Blood can be directly withdrawn through the venous cannula if the patient is under anesthesia.
	<ol>
		<li>Open the tube blister pack. Perform the venous puncture and fill the desired number of PRP tubes with whole blood. The vacuum within the tubes will enable automatic collection of the necessary volume of blood (~10 mL).</li>
		<li>Carefully turn the tubes upside down several times to mix the blood with anticoagulant.</li>
		<li>Inside a biosafety cabinet (class 2), remove 100 &#181;L of total whole blood using a 1 mL syringe for further blood cell number counts.</li>
	</ol>
	</li>
	<li>Centrifugation 
	NOTE: Ensure that the centrifuge is correctly balanced before starting it.
	<ol>
		<li>Once the blood is collected in the PRP tubes, if necessary, prepare a counterbalance tube (supplied separately) with water to the same level as the blood in the PRP tube. Place the filled tubes in the centrifuge opposite each other, ensuring that the machine is balanced.</li>
		<li>Centrifuge samples at 1,500 x g for 5 min. 
		NOTE: Set the corresponding rpm speed according to the centrifuge manufacturer&#8217;s instructions. After centrifugation, the blood will become fractionated. The red and white blood cells are trapped under the gel, and platelets settle on the surface of the gel (Figure 1).</li>
	</ol>
	</li>
	<li>Gently invert the PRP tube 20x to resuspend the platelet deposit in the plasma supernatant. 
	NOTE: Ensure that the platelets are fully detached from the gel. The plasma should change from clear and transparent to turbid. If platelet aggregates are present, they should be collected with the plasma.</li>
	<li>To collect PRP, take the plasma solution from the tube using a syringe transfer device connected to a 10 mL syringe. 
	NOTE: About 5 mL of PRP will be obtained from each tube. The PRP solution is now ready to mix in the final medium preparation. The solution can be kept at room temperature (RT) under the safety cabinet until the future steps involving hematology analyzer and use.</li>
	<li>Before using the PRP, determine the platelet concentration, mean platelet volume, and number of red and white blood cells using an automated hematology analyzer (Table of material). To do so, carefully withdraw 100 &#181;L of the solution and transfer into a 1.5 mL tube. 
	NOTE: PRP is used as an autologous culture supplement at a concentration between 5&#8211;20% v/v. The optimal concentration of PRP needs to be determined for each cell line. To prevent fibrin clot formation, the final culture media should be supplemented with 2 U/mL heparin.</li>
</ol>
2. Isolation of autologous fibroblasts and culture in FBS- or PRP-supplemented media
<ol>
	<li>Wash skin samples in phosphate-buffered saline (PBS) by gently shaking in a 50 mL polypropylene tube.</li>
	<li>If the skin sample is relatively large (&#62;10 cm2), place the sample on the lid of a 150 cm2 tissue culture dish (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. 
	NOTE: Use smaller tissue culture dish for smaller samples.</li>
	<li>When fat trimming is complete, cut the tissue into approximately 0.5 cm x 1.5 cm strips using a sterile scalpel.</li>
	<li>Add 5 mL of collagenase-dispase mix (Table of Materials; 14 W&#252;nsch units/mL) to a sterile 15 mL tube.</li>
	<li>Transfer the cut tissue to the tube, ensuring that the tissue pieces are submerged in the solution. Cap the tube securely.</li>
	<li>Place the tubes in an incubator at 37 &#176;C with orbital shaking for 150 min. After incubation, place the tube containing the tissue in the biosafety cabinet.</li>
	<li>Place the lid of a sterile 100 mm culture dish upside down in the biosafety cabinet. Transfer the digested tissue solution to the bottom of the 100 mm culture dish, without splashing. If any pieces of tissue remain in the tube, use a sterile 1 mL pipette or sterile forceps to transfer the tissue pieces to the bottom of the 100 mm culture dish.</li>
	<li>With the epidermis strip facing up, separate the intact epidermis sheet from the dermis by using two pairs of forceps. Hold the dermis of the tissue strip with a pair of forceps and the edge of the epidermis with another pair of forceps. Peel the dermis and epidermis apart in two pieces, keeping the separated pieces on the same lid. Try to perform this step quickly and repeat the manipulation for each piece of tissue.</li>
	<li>Transfer the dermal pieces to a new 100 mm culture dish containing PBS.</li>
	<li>Using laboratory forceps, place the dermal pieces in a 15 mL polypropylene tube with 3 mL of 0.3% trypsin/PBS. Incubate for 10-20 min in a 37 &#176;C water bath and invert the tube several times every 2&#8722;3 min.</li>
	<li>To stop the enzymatic reaction, add 3&#8722;5 mL of ice-cold complete growth medium (Dulbecco&#8217;s modified Eagle medium [DMEM] or RPMI containing 10% FBS). Vortex the tube vigorously several times. Filter the fibroblast suspension through an 85 &#956;m nylon mesh (placed over the top of a 50 mL tube) to remove dermal debris.</li>
	<li>Centrifuge for 10 min at 150 x g, at 4 &#176;C. Aspirate the supernatant and resuspend the pellet in 100&#8722;1,000 &#956;L of complete growth medium.</li>
	<li>Using trypan blue (dilution factor of x2) mixed with the cell suspension, count the number of total and viable cells by filling the chamber of the haemocytometer. 
	NOTE: Cell viability depends on the conditions used for enzymatic digestion. To increase cell recovery and cell viability, the skin sample may be cut into smaller pieces.</li>
	<li>Plate 3&#8722;10 x 104 cells in 5 mL of complete FBS growth medium (DMEM, 10% FBS heat-inactivated for 60 min at 56 &#176;C, 1% 1 M HEPES buffer solution, 1% 100x nonessential amino acid mixture, 1% 100x L-glutamine, 1% 100x penicillin/streptomycin, 1% 100x sodium pyruvate) in a 25 cm2 tissue culture flask. Incubate at 37 &#176;C. 
	NOTE: Viable fibroblasts will attach to the flask within 24 h and begin to exhibit the spindle-shape in 2&#8722;3 days.</li>
	<li>On day 2, aspirate the medium containing nonadherent cells and add fresh medium. 
	NOTE: As dead cells in the culture medium affect the growth of viable fibroblasts, nonadherent, dead cells must be removed from the culture.</li>
	<li>Change the medium every 3&#8722;4 days until the culture reaches 70&#8722;80% confluency.</li>
	<li>To harvest fibroblasts, wash with PBS and incubate with trypsin/EDTA solution for 3 min at 37 &#176;C in the incubator.</li>
	<li>To stop the reaction, add 3&#8722;5 mL of warm complete growth medium (DMEM or RPMI containing 10% FBS). Take an aliquot of the cell suspension to count the cells with the haemocytometer. Centrifuge the suspension for 5 min at 200 x g and remove the medium by aspiration.</li>
	<li>Culture the fibroblast in PRP medium (DMEM, 20% PRP, 1% 1 M HEPES buffer solution, 1% 100x nonessential amino acid mixture, 1% 100x L-glutamine, 1% 100x penicillin/streptomycin, 1% 100x sodium pyruvate, 2 U/mL heparin) in the incubator at 37 &#176;C. 
	NOTE: The minimum cell density recommended is 4,000 viable cells/cm2.</li>
	<li>To assess PRP effects on fibroblast proliferation, seed fibroblasts (passage 2) in 24 well plates at a density of 8 x 103 cells per well in PRP medium with different PRP concentrations (1%, 5%, 10%, 20%, 30%, 40%, and 50%) and with 2 U/mL heparin or under classical culture medium conditions (10% FBS). After 7 days of culture, add a vital dye (cell proliferation violet) to cells and assess the proliferation by flow cytometry.</li>
	<li>To study cytoskeletal rearrangements, seed cells in 96 well black/clear flat bottom plates at a concentration of 1 x 105 cells/mL in complete FBS growth DMEM (see step 2.14) supplemented with 0.5% FBS for 24 h. Treat cells with 10% FBS or different PRP concentrations (1%, 5%, 10%, 20%, 30%, 40%, and 50%) for 7 days.&#160;
	<ol>
		<li>Fix the fibroblasts with 4% paraformaldehyde for 10 min and permeabilize with 0.1% Triton X-100 for 5 min at RT. Stain the fibroblasts with 50 mL of 5 U/mL phalloidin, wash 2x with PBS, and mark with 50 mL of 1 mg/mL 4&#8242;,6-diamidino-2-phenylindole (DAPI) for 5 min.&#160;Cytation 3 cell imaging multimode reader (BioTek) was used to visualize phalloidin staining.</li>
	</ol>
	</li>
</ol>

<ol><li>Kumar, S., Mahajan, B. B., Kaur, S., Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autologous+therapies+in+dermatology%5BTitle%5D)+AND+%22The+Journal+of+Clinical+and+Aesthetic+Dermatology%22%5BJournal%5D)">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/pubmed?term=((The+survey+on+cellular+and+engineered+tissue+therapies+in+Europe+in+2009%5BTitle%5D)+AND+%22Tissue+Engineering.+Part+A%22%5BJournal%5D)">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/pubmed?term=((Dermal+fibroblasts-A+heterogeneous+population+with+regulatory+function+in+wound+healing%5BTitle%5D)+AND+%22Cytokine+%26+Growth+Factor+Reviews%22%5BJournal%5D)">Dermal fibroblasts-A heterogeneous population with regulatory function in wound healing.</a> Cytokine & 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/pubmed?term=((Alteration+of+skin+properties+with+autologous+dermal+fibroblasts%5BTitle%5D)+AND+%22International+Journal+of+Biological+Sciences%22%5BJournal%5D)">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/pubmed?term=((Fibroblasts+as+maestros+orchestrating+tissue+regeneration%5BTitle%5D)+AND+%22Journal+of+Tissue+Engineering+and+Regenerative+Medicine%22%5BJournal%5D)">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/pubmed?term=((Autologous+cell+therapy%3A+will+it+replace+dermal+fillers%5BTitle%5D)+AND+%22Facial+Plastic+Surgery+Clinics+of+North+America%22%5BJournal%5D)">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/pubmed?term=((Fibroblasts+as+a+practical+alternative+to+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Journal+of+Translational+Medicine%22%5BJournal%5D)">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/pubmed?term=((Alternatives+to+the+use+of+fetal+bovine+serum%3A+serum-free+cell+culture%5BTitle%5D)+AND+%22ALTEX%22%5BJournal%5D)">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/pubmed?term=((A+consensus+introduction+to+serum+replacements+and+serum-free+media+for+cellular+therapies%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">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/pubmed?term=((Fetal+Bovine+Serum+%28FBS%29%3A+Past+-+Present+-+Future%5BTitle%5D)+AND+%22ALTEX%22%5BJournal%5D)">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/pubmed?term=((Platelet-Rich+Plasma%3A+The+Choice+of+Activation+Method+Affects+the+Release+of+Bioactive+Molecules%5BTitle%5D)+AND+%22BioMed+Research+International%22%5BJournal%5D)">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/pubmed?term=((Platelet-Rich+Plasma+for+Skin+Rejuvenation%3A+Facts%2C+Fiction%2C+and+Pearls+for+Practice%5BTitle%5D)+AND+%22Facial+Plastic+Surgery+Clinics+of+North+America%22%5BJournal%5D)">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/pubmed?term=((Review+of+concentration+yields+in+commercially+available+platelet-rich+plasma+%28PRP%29+systems%3A+a+call+for+PRP+standardization%5BTitle%5D)+AND+%22Regional+Anesthesia+and+Pain+Medicine%22%5BJournal%5D)">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/pubmed?term=((Autologous+Platelet-Rich+Plasma+%28CuteCell+PRP%29+Safely+Boosts+In+Vitro+Human+Fibroblast+Expansion%5BTitle%5D)+AND+%22Tissue+Engineering.+Part+A%22%5BJournal%5D)">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/pubmed?term=((Preclinical+safety+studies+on+autologous+cultured+human+skin+fibroblast+transplantation%5BTitle%5D)+AND+%22Cell+Transplantation%22%5BJournal%5D)">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/pubmed?term=((Efficacy+of+Autologous+Cultured+Fibroblast+Cells+as+a+Treatment+for+Patients+with+Facial+Contour+Defects%3A+A+Clinical+Replication+Study%5BTitle%5D)+AND+%22Journal+of+Cosmetics%2C+Dermatological+Sciences+and+Applications%22%5BJournal%5D)">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/pubmed?term=((Autologous+fibroblast+culture+in+the+repair+of+aging+skin%5BTitle%5D)+AND+%22Dermatologic+Surgery%22%5BJournal%5D)">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/pubmed?term=((Autologous+fibroblast+suspension+for+the+treatment+of+refractory+diabetic+foot+ulcer%5BTitle%5D)+AND+%22Indian+Journal+of+Dermatology%2C+Venereology+and+Leprology%22%5BJournal%5D)">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/pubmed?term=((Human+Platelet+Lysate+as+a+Functional+Substitute+for+Fetal+Bovine+Serum+in+the+Culture+of+Human+Adipose+Derived+Stromal%2FStem+Cells%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">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/pubmed?term=((Autologous+platelet-rich+plasma%3A+a+biological+supplement+to+enhance+adipose-derived+mesenchymal+stem+cell+expansion%5BTitle%5D)+AND+%22Tissue+Engineering+Part+C%3A+Methods%22%5BJournal%5D)">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/pubmed?term=((Autologous+platelet-rich+plasma+for+treating+chronic+wounds%5BTitle%5D)+AND+%22The+Cochrane+Database+of+Systematic+Reviews%22%5BJournal%5D)">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/pubmed?term=((Use+of+platelet-rich+plasma+and+hyaluronic+acid+in+the+loss+of+substance+with+bone+exposure%5BTitle%5D)+AND+%22Advances+in+Skin+%26+Wound+Care%22%5BJournal%5D)">Use of platelet-rich plasma and hyaluronic acid in the loss of substance with bone exposure.</a> Advances in Skin & Wound Care. 24 (4), 176-181 (2011).</li>
<li>Nicoli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Severe+hidradenitis+suppurativa+treatment+using+platelet-rich+plasma+gel+and+Hyalomatrix%5BTitle%5D)+AND+%22International+Wound+Journal%22%5BJournal%5D)">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/pubmed?term=((Application+of+platelet-rich+plasma+in+maxillofacial+surgery%3A+clinical+evaluation%5BTitle%5D)+AND+%22The+Journal+of+Craniofacial+Surgery%22%5BJournal%5D)">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/pubmed?term=((Adjunctive+Platelet-Rich+Plasma+%28PRP%29+in+Infrabony+Regenerative+Treatment%3A+A+Systematic+Review+and+RCT%27s+Meta-Analysis%5BTitle%5D)+AND+%22Stem+Cells+International%22%5BJournal%5D)">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/pubmed?term=((Autologous+pure+platelet-rich+plasma+injections+for+facial+skin+rejuvenation%3A+Biometric+instrumental+evaluations+and+patient-reported+outcomes+to+support+antiaging+effects%5BTitle%5D)+AND+%22Journal+of+Cosmetic+Dermatology%22%5BJournal%5D)">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/pubmed?term=((Magnetic+resonance+imaging+and+ultrasound+evaluation+after+breast+autologous+fat+grafting+combined+with+platelet-rich+plasma%5BTitle%5D)+AND+%22Plastic+and+Reconstructive+Surgery%22%5BJournal%5D)">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/pubmed?term=((Breast+Reconstruction+with+Enhanced+Stromal+Vascular+Fraction+Fat+Grafting%3A+What+Is+the+Best+Method%5BTitle%5D)+AND+%22Plastic+and+Reconstructive+Surgery%22%5BJournal%5D)">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/pubmed?term=((Platlet+Rich+Plasma+%28PRP%29+Improves+Fat+Grafting+Outcomes%5BTitle%5D)+AND+%22World+Journal+of+Plastic+Surgery%22%5BJournal%5D)">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/pubmed?term=((Culture+Medium+Supplements+Derived+from+Human+Platelet+and+Plasma%3A+Cell+Commitment+and+Proliferation+Support%5BTitle%5D)+AND+%22Frontiers+in+Bioengineering+and+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((The+effect+of+heparin+on+cell+proliferation+and+type-I+collagen+synthesis+by+adult+human+dermal+fibroblasts%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta%22%5BJournal%5D)">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/pubmed?term=((Hypersensitivity+reactions+to+heparins%5BTitle%5D)+AND+%22Current+Opinion+in+Allergy+and+Clinical+Immunology%22%5BJournal%5D)">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-Cuénod, B., Modarressi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Platelet+Rich+Plasma+Promotes+Proliferation+of+Adipose+Derived+Mesenchymal+Stem+Cells+via+Activation+of+AKT+and+Smad2+Signaling+Pathways%5BTitle%5D)+AND+%22Stem+Cell+Research+%26+Therapy%22%5BJournal%5D)">Platelet Rich Plasma Promotes Proliferation of Adipose Derived Mesenchymal Stem Cells via Activation of AKT and Smad2 Signaling Pathways.</a> Stem Cell Research & Therapy. 5 (8), (2015).</li>
</ol>

This project has been funded by Regen Lab SA. Sarah Berndt is the cell therapy head project manager for Regen Lab and is employed by Regen Lab SA. Antoine Turzi is the CEO of RegenLab.

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

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...

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 ...

production-autologous-platelet-rich-plasma-for-boosting-vitro-human

Research

JoVE Journal

Bioengineering

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

This video demonstrates a technique for the differentiation of megakaryocytes and the formation of platelets. Hematopoietic stem progenitor cells — isolated from human umbilical cord blood — differentiate into mature megakaryocytes in the presence of thrombopoietin. The mature megakaryocytes produce cytoplasmic extensions termed proplatelets, which give rise to individual platelets.

An In Vitro Method for the Differentiation of Megakaryocytes and Platelet Formation

Source:Sathish Muthu1,2,31Orthopaedic Research Group, Coimbatore.2Department of Biotechnology, Faculty of Engineering, Karpagam Academy of Higher Education.3Department of Orthopaedics, Government Medical College.This video demonstrates the preparation of high-quality Platelet-Rich Plasma (PRP) by screening for platelet count, collecting and centrifuging blood, and isolating platelets. This PRP is used in therapeutic applications for tissue regeneration and healing.

Preparation of Platelet-Rich Plasma from Human Peripheral Blood

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.

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

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.

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Platelet-rich plasma (PRP) is used in the treatment of equine musculoskeletal disorders. Different techniques yield PRP with varying platelet and leukocyte concentrations. This article describes a method for obtaining PRP from horse blood.

A Simple Double Centrifugation Tube Method to Obtain Platelet-rich Plasma from Equine Blood

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.

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

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.

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

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.

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

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).

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

Learn to produce autologous platelet-rich plasma for boosting human fibroblast expansion in vitro. This standardized five-minute protocol enables safe, massive cell growth for regenerative medicine applications.

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

Explore More Videos